| United States Patent | 7,582,808 |
| Dhugga , et al. | September 1, 2009 |
Cellulose synthase encoding nucleic acids and uses thereof
Abstract
The invention provides isolated cellulose synthase nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering cellulose synthase concentration and/or composition of plants. The invention further provides recombinant expression cassettes, host cells, and transgenic plants.
| Inventors: | Dhugga; Kanwarpal S (Johnston, IA), Helentjaris; Timothy G (Tucson, AZ), Bowen; Benjamin A (Berkeley, CA), Wang; Xun (Chapel Hill, NC) |
| Assignee: |
Pioneer Hi-Bred International, Inc.
(Johnston,
IA)
|
| Appl. No.: | 11/867,246 |
| Filed: | October 4, 2007 |
| Application Number | Filing Date | Patent Number | Issue Date | ||
| 11493187 | Jul., 2006 | 7312377 | |||
| 10961254 | Oct., 2004 | 7214852 | |||
| 10160719 | Jun., 2002 | 6803498 | |||
| 09371383 | Aug., 1999 | ||||
| 60096822 | Aug., 1998 | ||||
| Current U.S. Class: | 800/284 ; 435/430.1; 435/468; 435/69.1; 800/278; 800/286; 800/290; 800/295; 800/298; 800/317; 800/320 |
| Current International Class: | C12N 15/29 (20060101); A01H 1/00 (20060101); C12N 15/82 (20060101); A01H 5/10 (20060101) |
| 5723764 | March 1998 | Nichols |
| 9800549 | Jan., 1998 | WO | |||
| 9818949 | May., 1998 | WO | |||
| 0004168 | Jan., 2000 | WO | |||
Amor, Y., et al.; "A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants"; PNAS (Sep. 1995) 92:9353-9357; National Academy of Sciences; Washington, DC US. cited by other . Haigler, C., et al.; "New hope for old dreams: Evidence that plant cellulose synthase genes have finally been identified"; PNAS (Oct. 1996) 93:12082-12085; National Academy of Sciences; Washington, DC US. cited by other . Pear, J.R., et al.; "Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase"; PNAS (Oct. 1996) 93:12637-12642; National Academy of Sciences; Washington, DC US. cited by other . Arioli, T., et al.; "Molecular analysis of cellulose biosynthesis in Arabidopsis"; Science (Jan. 1998) 279:717-720; American Association for the Advancement of Science; Washington, DC US. cited by other . AF027174; "A. thaliana cellulose synthase catalytic subunit EMBL/GenBank/DDBJ database entry" (Feb. 1999). cited by other . AC048947; "A. thaliana cellulose synthase catalytic subunit EMBL database entry" (Jun. 1998). cited by other . Amor, Y., et al.; "Evidence for a cyclic diguanylic acid-dependent cellulose synthase in plants"; Plant Cell (1991) 3(9):989-995; American Society of Plant Physiologists; Rockville, MD US. cited by other . Delmer, D.; "Cellulose Biosynthesis: Exciting Times for a Difficult Field of Study"; Ann. Rev. Plant Physiol. Plant Mol. Bio. (1999) 50:245-276; Springer; The Netherlands. cited by other . Arioli, et al.; "Accession No. AC048948, Molecular Analysis of Cellulose Biosynthesis in Arabidopsis"; Science (1998) 279:717-720 (XP-002140697); American Association for the Advancement of Science; Washington, DC US. cited by other . Arioli, et al.; "Accession No. AF030052, Molecular Analysis of Cellulose Biosynthesis in Arabidopsis"; Science (1998) 279:717-720 (XP-002140698); American Association for the Advancement of Science; Washington, DC US. cited by other . Arioli, et al.; "Accession No. AC048946, Molecular Analysis of Cellulose Biosynthesis in Arabidopsis"; Science (1998) 279:717-720 (XP-002140699); American Association for the Advancement of Science; Washington, DC US. cited by other . Arioli, et al.; "Accession No. AF027173, Molecular Analysis of Cellulose Biosynthesis in Arabidopsis" Science (1998) 279:717-720 (XP-00214700); American Association for the Advancement of Science; Washington, DC US. cited by other . Wu, et al.; "Accession No. AC065338"; (1998) (XP-002140701). cited by other. |
Primary Examiner: Ibrahim; Medina A
CROSS REFERENCE TO RELATED APPLICATION
This divisional application claims priority to and hereby incorporates by reference, application Ser. No. 11/493,187 filed Jul. 26, 2006, now granted as U.S. Pat. No. 7,312,377, which was a divisional of application Ser. No. 10/961,254 filed Oct. 8, 2004, now granted as U.S. Pat. No. 7,214,852, which was a divisional of application Ser. No. 10/160,719 filed Jun. 3, 2002, now granted as U.S. Pat. No. 6,803,498, which was a continuation of non-provisional application 09/371,383 filed Aug. 6, 1999, now abandoned, and provisional application 60/096,822 filed Aug. 17, 1998.
What is claimed is:
1. An isolated nucleic acid encoding a functional cellulose synthase, said polynucleotide selected from the group consisting of: (a) a polynucleotide having at least 95% sequence identity, as determined by the BLAST 2.0 algorithm under default parameters, to the full length polypeptide sequence of SEQ ID NO: 2; (b) a polynucleotide encoding a polypeptide of SEQ ID NO: 2; and (c) a polynucleotide of SEQ ID NO: 1.
2. A recombinant expression cassette, comprising the polynucleotide of claim 1 operably linked, in sense or anti-sense orientation, to a promoter.
3. A host cell comprising the recombinant expression cassette of claim 2.
4. A transgenic plant comprising a recombinant expression cassette of claim 2.
5. The transgenic plant of claim 4, wherein the plant is a monocot.
6. The transgenic plant of claim 4, wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet.
7. A transgenic seed from the transgenic plant of claim 4.
8. A method of modulating the level of cellulose synthase in a plant cell, comprising: (a) transforming the plant cell with a recombinant expression cassette comprising the polynucleotide of claim 1 operably linked to a promoter; and (b) culturing the transformed plant cell under plant cell growing conditions; wherein the level of cellulose synthase in said transformed plant cell is modulated.
9. The method of claim 8, further comprising regenerating a plant from the transformed plant cell.
10. The method of claim 9, wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet.
11. The method of claim 8, wherein the promoter is a tissue-preferred promoter.
12. The method of claim 8, wherein the level of cellulose synthase is increased.
13. The method of claim 8 wherein the polynucleotide encoding a functional cellulose synthase is SEQ ID NO: 1.
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
Polysaccharides constitute the bulk of the plant cell walls and have been traditionally classified into three categories: cellulose, hemicellulose, and pectin. Fry, (1988), The growing plant cell wall: Chemical and metabolic analysis. New York: Longman Scientific & Technical. Whereas cellulose is made at the plasma membrane and directly laid down into the cell wall, hemicellulosic and pectic polymers are first made in the Golgi apparatus and then exported to the cell wall by exocytosis. Ray, et al., (1976) Ber. Deutsch. Bot. Ges. Bd. 89:121-146. The variety of chemical linkages in the pectic and hemicellulosic polysaccharides indicates that there must be tens of polysaccharide synthases in the Golgi apparatus. Darvill, et al., (1980). The primary cell walls of flowering plants. In The Plant Cell (N. E. Tolbert, ed.), Vol. 1 in Series: The biochemistry of plants: A comprehensive treatise, eds. P. K. Stumpf and E. E. Conn (New York: Academic Press), pp. 91-162.
Cellulose, by virtue of its ability to form semicrystalline microfibrils, has a very high tensile strength which approaches that of some metals. Niklas, (1992) Plant Biomechanics: An engineering approach to plant form and function, The University of Chicago Press, pp. 607. Bending strength of the culm of normal and brittle-culm mutants of barley has been found to be directly correlated with the concentration of cellulose in the cell wall. Kokubo, et al., (1989) Plant Physiology 91:876-882; Kokubo, et al., (1991) Plant Physiology 97:509-514.
Even though sugar and polysaccharide compositions of the plant cell walls have been well characterized, very limited progress has been made toward identification of the enzymes involved in polysaccharides formation, the reason being their labile nature and recalcitrance to solubilization by available detergents. Sporadic claims for the identification of cellulose synthase from plant sources have been made over the years. Callaghan and Benziman, (1984), Nature 311:165-167; Okuda, et al., (1993) Plant Physiol. 101:1131-1142. However, these claims have been met with skepticism. Callaghan and Benziman, (1985) Nature 314:383-384; Delmer, et al., (1993) Plant Physiol. 103:307-308. It was only recently that a putative gene for plant cellulose synthase (CelA) was cloned from the developing cotton fibers based on homology to the bacterial gene. Pear, et al., Proc. Natl. Acad. Sci. (USA) 93:12637-12642; Saxena, et al., (1990), Plant Molecular Biology 15:673-684; see also, WO 9818949.
As brittle snap is a major problem in corn breeding, what is needed in the art are compositions and methods for manipulating cellulose concentration in the cell wall and thereby altering plant stalk quality for improved standability or silage. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to cellulose synthases. It is an object of the present invention to provide: 1) nucleic acids and proteins relating to maize cellulose synthases; 2) transgenic plants comprising the nucleic acids of the present invention; 3) methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA or RNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by an isolated nucleic acid of the present invention.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of specified length which selectively hybridizes under stringent conditions to a polynucleotide of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In yet another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide, the polynucleotide having a specified sequence identity to an identical length of a nucleic acid of the present invention or a complement thereof.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide having a sequence of a nucleic acid amplified from a Zea mays nucleic acid library using at least two primers or their complements, one of which selectively hybridizes under stringent conditions to a locus of the nucleic acid comprising the 5' terminal coding region and the other primer selectively hybridizing, under stringent conditions, to a locus of the nucleic acid comprising the 3' terminal coding region, and wherein both primers selectively hybridize within the coding region. In some embodiments, the nucleic acid library is a cDNA library.
In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid, wherein the nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to a host cell transfected with this recombinant expression cassette. In some embodiments, the present invention relates to a protein of the present invention which is produced from this host cell.
In a further aspect, the present invention relates to a heterologous promoter operably linked to a non-isolated polynucleotide of the present invention, wherein the polypeptide is encoded by a nucleic acid amplified from a nucleic acid library.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. In some embodiments, the transgenic plant is Zea mays. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of modulating expression of the genes encoding the proteins of the present invention in a plant cell capable of plant regeneration, comprising the steps of (a) transforming a plant cell with a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; (b) growing the plant cell under plant growing conditions; and (c) inducing expression of the polynucleotide for a time sufficient to modulate expression of the genes in the plant. In some embodiments, the plant is maize. Expression of the genes encoding the proteins of the present invention can be increased or decreased relative to a non-transformed control plant.
Definitions
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and electronics Terms (5.sup.th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing, et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
The term "antibody" includes reference to antigen binding forms of antibodies (e.g., Fab, F(ab).sub.2). The term "antibody" frequently refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). However, while various antibody fragments can be defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments such as single chain Fv, chimeric antibodies (i.e., comprising constant and variable regions from different species), humanized antibodies (i.e., comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies (e.g., bispecific antibodies).
The term "antigen" includes reference to a substance to which an antibody can be generated and/or to which the antibody is specifically immunoreactive. The specific immunoreactive sites within the antigen are known as epitopes or antigenic determinants. These epitopes can be a linear array of monomers in a polymeric composition--such as amino acids in a protein--or consist of or comprise a more complex secondary or tertiary structure. Those of skill will recognize that all immunogens (i.e., substances capable of eliciting an immune response) are antigens; however some antigens, such as haptens, are not immunogens but may be made immunogenic by coupling to a carrier molecule. An antibody immunologically reactive with a particular antigen can be generated in vivo or by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors. See, e.g., Huse, et al., (1989) Science 246:1275-1281; and Ward, et al., (1989) Nature 341:544-546; and Vaughan, et al., (1996) Nature Biotech. 14:309-314.
As used herein, "antisense orientation" includes reference to a duplex polynucleotide sequence which is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
As used herein, "chromosomal region" includes reference to a length of a chromosome which may be measured by reference to the linear segment of DNA which it comprises. The chromosomal region can be defined by reference to two unique DNA sequences, i.e., markers.
The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and incorporated herein by reference.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the native protein for it's native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins W.H. Freeman and Company.
By "encoding" or "encoded", with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as are present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolumn (Proc. Natl. Acad. Sci. (USA) 82:2306-2309 (1985)), or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.
When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray, et al., (1989) Nucl. Acids Res. 17:477-498). Thus, the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants are listed in Table 4 of Murray, et al., above.
As used herein "full-length sequence" in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of, a native (non-synthetic), endogenous, catalytically active form of the specified protein. Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extension, S1 protection, and ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention. Additionally, consensus sequences typically present at the 5' and 3' untranslated regions of mRNA aid in the identification of a polynucleotide as full-length. For example, the consensus sequence ANNNNAUGG, where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5' end. Consensus sequences at the 3' end, such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3' end.
The term "gene activity" refers to one or more steps involved in gene expression, including transcription, translation, and the functioning of the protein encoded by the gene.
As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
By "host cell" is meant a cell which contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells. A particularly preferred monocotyledonous host cell is a maize host cell.
The term "hybridization complex" includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
By "immunologically reactive conditions" or "immunoreactive conditions" is meant conditions which allow an antibody, generated to a particular epitope, to bind to that epitope to a detectably greater degree (e.g., at least 2-fold over background) than the antibody binds to substantially all other epitopes in a reaction mixture comprising the particular epitope. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols. See Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions.
The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The terms "isolated" refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a locus in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. The alteration to yield the synthetic material can be performed on the material within or removed from its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by non-natural, synthetic (i.e., "man-made") methods performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling, et al., PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids which are "isolated" as defined herein, are also referred to as "heterologous" nucleic acids.
Unless otherwise stated, the term "cellulose synthase nucleic acid" is a nucleic acid of the present invention and means a nucleic acid comprising a polynucleotide of the present invention (a "cellulose synthase polynucleotide") encoding a cellulose synthase polypeptide. A "cellulose synthase gene" is a gene of the present invention and refers to a non-heterologous genomic form of a full-length cellulose synthase polynucleotide.
As used herein, "localized within the chromosomal region defined by and including" with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers.
As used herein, "marker" includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A "polymorphic marker" includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes in that pair to be followed. A genotype may be defined by use of one or a plurality of markers.
As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
By "nucleic acid library" is meant a collection of isolated DNA or RNA molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook, et al., Molecular Cloning--A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel, et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).
As used herein "operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
As used herein, the term "plant" includes reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same. Plant cell, as used herein includes, without limitation, cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants include maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet.
As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Exemplary modifications are described in most basic texts, such as, Proteins--Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pp. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter, et al., (1990) Meth. Enzymol. 182:626-646 and Rattan, et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli or other cells, prior to proteolytic processing, almost invariably will be N-formylmethionine. During post-translational modification of the peptide, a methionine residue at the NH.sub.2-terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.
As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as "tissue preferred". Promoters which initiate transcription only in certain tissue are referred to as "tissue specific". A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which is active under most environmental conditions.
The term "cellulose synthase polypeptide" is a polypeptide of the present invention and refers to one or more amino acid sequences, in glycosylated or non-glycosylated form. The term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof. A "cellulose synthase protein" is a protein of the present invention and comprises a cellulose synthase polypeptide.
As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term "recombinant" as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
As used herein, a "recombinant expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
The term "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.
The term "specifically reactive", includes reference to a binding reaction between an antibody and a protein having an epitope recognized by the antigen binding site of the antibody. This binding reaction is determinative of the presence of a protein having the recognized epitope amongst the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to an analyte having the recognized epitope to a substantially greater degree (e.g., at least 2-fold over background) than to substantially all other analytes lacking the epitope which are present in the sample.
The terms "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times. SSC (20.times. SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times. SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times. SSC at 60 to 65.degree. C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
As used herein, "transgenic plant" includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
As used herein, "vector" includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".
(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, "comparison window" means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482; by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, (1988) Gene 73:237-244; Higgins and Sharp, (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Research 16:10881-90; Huang, et al., (1992) Computer Applications in the Biosciences 8:155-65, and Pearson, et al., (1994) Methods in Molecular Biology 24:307-331. The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul, et al., (1997) Nucleic Acids Res. 25:3389-3402. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem., 17:149-163) and XNU (Claverie and States, (1993) Comput. Chem., 17:191-201) low-complexity filters can be employed alone or in combination.
(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci., 4:11-17 e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
(d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
(e) (i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90% and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
(e) (ii) The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
DETAILED DESCRIPTION OF THE INVENTION
Overview
The present invention provides, among other things, compositions and methods for modulating (i.e., increasing or decreasing) the level of polypeptides of the present invention in plants. In particular, the polypeptides of the present invention can be expressed at developmental stages, in tissues, and/or in quantities which are uncharacteristic of non-recombinantly engineered plants. Thus, the present invention provides utility in such exemplary applications as improvement of stalk quality for improved stand or silage. Further, the present invention provides for an increased concentration of cellulose in the pericarp; hardening the kernel and thus improving its handling ability.
The present invention also provides isolated nucleic acid comprising polynucleotides of sufficient length and complementarity to a gene of the present invention to use as probes or amplification primers in the detection, quantitation, or isolation of gene transcripts. For example, isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for desired transgenic plants, for detecting mutations in the gene (e.g., substitutions, deletions, or additions), for monitoring upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allelic variants (polymorphisms) of the gene, or for use as molecular markers in plant breeding programs. The isolated nucleic acids of the present invention can also be used for recombinant expression of their encoded polypeptides, or for use as immunogens in the preparation and/or screening of antibodies. The isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more genes of the present invention in a host cell, tissue, or plant. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation.
The present invention also provides isolated proteins comprising a polypeptide of the present invention (e.g., preproenzyme, proenzyme, or enzymes). The present invention also provides proteins comprising at least one epitope from a polypeptide of the present invention. The proteins of the present invention can be employed in assays for enzyme agonists or antagonists of enzyme function, or for use as immunogens or antigens to obtain antibodies specifically immunoreactive with a protein of the present invention. Such antibodies can be used in assays for expression levels, for identifying and/or isolating nucleic acids of the present invention from expression libraries, or for purification of polypeptides of the present invention.
The isolated nucleic acids and proteins of the present invention can be used over a broad range of plant types, particularly monocots such as the species of the Family Graminiae including Sorghum bicolor and Zea mays. The isolated nucleic acid and proteins of the present invention can also be used in species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Triticum, Bambusa, Dendrocalamus, and Melocanna.
Nucleic Acids
The present invention provides, among other things, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising a polynucleotide of the present invention.
A polynucleotide of the present invention is inclusive of:
(a) a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54 and 58, and conservatively modified and polymorphic variants thereof, including exemplary polynucleotides of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53 and 57;
(b) a polynucleotide which is the product of amplification from a Zea mays nucleic acid library using primer pairs which selectively hybridize under stringent conditions to loci within a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53 and 57, wherein the polynucleotide has substantial sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53 and 57;
(c) a polynucleotide which selectively hybridizes to a polynucleotide of (a) or (b);
(d) a polynucleotide having a specified sequence identity with polynucleotides of (a), (b) or (c);
(e) a polynucleotide encoding a protein having a specified number of contiguous amino acids from a prototype polypeptide, wherein the protein is specifically recognized by antisera elicited by presentation of the protein and wherein the protein does not detectably immunoreact to antisera which has been fully immunosorbed with the protein;
(f) complementary sequences of polynucleotides of (a), (b), (c), (d) or (e); and
(g) a polynucleotide comprising at least a specific number of contiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e) or (f).
A. Polynucleotides Encoding a Polypeptide of the Present Invention or Conservatively Modified or Polymorphic Variants Thereof
As indicated in (a), above, the present invention provides isolated nucleic acids comprising a polynucleotide of the present invention, wherein the polynucleotide encodes a polypeptide of the present invention, or conservatively modified or polymorphic variants thereof. Those of skill in the art will recognize that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. Such "silent variations" can be used, for example, to selectively hybridize and detect allelic variants of polynucleotides of the present invention. Accordingly, the present invention includes polynucleotides of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53 and 57, and silent variations of polynucleotides encoding a polypeptide of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54 and 58. The present invention further provides isolated nucleic acids comprising polynucleotides encoding conservatively modified variants of a polypeptide of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54 and 58. Additionally, the present invention further provides isolated nucleic acids comprising polynucleotides encoding one or more polymorphic (allelic) variants of polypeptides/polynucleotides. Polymorphic variants are frequently used to follow segregation of chromosomal regions in, for example, marker assisted selection methods for crop improvement.
B. Polynucleotides Amplified from a Zea mays Nucleic Acid Library
As indicated in (b) above, the present invention provides an isolated nucleic acid comprising a polynucleotide of the present invention, wherein the polynucleotides are amplified from a Zea mays nucleic acid library. Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23, and Mo17 are known and publicly available. Other publicly known and available maize lines can be obtained from the Maize Genetics Cooperation (Urbana, Ill.). The nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing. cDNA libraries can be normalized to increase the representation of relatively rare cDNAs. In optional embodiments, the cDNA library is constructed using a full-length cDNA synthesis method. Examples of such methods include Oligo-Capping (Maruyama and Sugano, (1994) Gene 138:171-174), Biotinylated CAP Trapper (Carninci, et al., (1996) Genomics 37:327-336), and CAP Retention Procedure (Edery, et al., (1995) Molecular and Cellular Biology 15:3363-3371). cDNA synthesis is often catalyzed at 50-55.degree. C. to prevent formation of RNA secondary structure. Examples of reverse transcriptases that are relatively stable at these temperatures are SuperScript II Reverse Transcriptase (Life Technologies, Inc.), AMV Reverse Transcriptase (Boehringer Mannheim) and RetroAmp Reverse Transcriptase (Epicentre). Rapidly growing tissues, or rapidly dividing cells are preferably used as mRNA sources such as from the elongating internode of corn plants.
The polynucleotides of the present invention include those amplified using the following primer pairs: SEQ ID NOS: 3 and 4 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 1; SEQ ID NOS: 7 and 8 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 5; and SEQ ID NOS: 11 and 12 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 9. SEQ ID NOS: 15 and 16 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 13. SEQ ID NOS: 19 and 20 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 17; SEQ ID NOS: 23 and 24 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 21; and SEQ ID NOS: 27 and 28 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 25. SEQ ID NOS: 31 and 32 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 29. SEQ ID NOS: 35 and 36 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 33; SEQ ID NOS: 39 and 40 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 37; and SEQ ID NOS: 43 and 44 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 41. SEQ ID NOS: 47 and 48 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 45. SEQ ID NOS: 51 and 52 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 49; SEQ ID NOS: 55 and 56 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 53; and SEQ ID NOS: 59 and 60 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 57.
The present invention also provides subsequences of the polynucleotides of the present invention. A variety of subsequences can be obtained using primers which selectively hybridize under stringent conditions to at least two sites within a polynucleotide of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it. Primers are chosen to selectively hybridize, under stringent hybridization conditions, to a polynucleotide of the present invention. Generally, the primers are complementary to a subsequence of the target nucleic acid which they amplify. As those skilled in the art will appreciate, the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired amplification conditions.
In optional embodiments, the primers will be constructed so that they selectively hybridize under stringent conditions to a sequence (or its complement) within the target nucleic acid which comprises the codon encoding the carboxy or amino terminal amino acid residue (i.e., the 3' terminal coding region and 5' terminal coding region, respectively) of the polynucleotides of the present invention. Optionally within these embodiments, the primers will be constructed to selectively hybridize entirely within the coding region of the target polynucleotide of the present invention such that the product of amplification of a cDNA target will consist of the coding region of that cDNA. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40 or 50 nucleotides in length. Those of skill will recognize that a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence. A non-annealing sequence at the 5' end of a primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.
The amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra. The resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention. Methods for protein synthesis from PCR derived templates are known in the art and available commercially. See, e.g., Amersham Life Sciences, Inc, Catalog '97, p. 354.
Methods for obtaining 5' and/or 3' ends of a vector insert are well known in the art. See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in Frohman, M. A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc., San Diego, 1990), pp. 28-38); see also, U.S. Pat. No. 5,470,722, and Current Protocols in Molecular Biology, Unit 15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Frohman and Martin, (1989) Techniques 1:165.
C. Polynucleotides which Selectively Hybridize to a Polynucleotide of (A) or (B)
As indicated in (c), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide of paragraphs (A) or (B) as discussed, above. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising the polynucleotides of (A) or (B). For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated or otherwise complementary to a cDNA from a dicot or monocot nucleic acid library. Exemplary species of monocots and dicots include, but are not limited to: corn, canola, soybean, cotton, wheat, sorghum, sunflower, oats, sugar cane, millet, barley, and rice. Optionally, the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA libraries can be normalized to increase the representation of rare sequences. Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.
D. Polynucleotides Having a Specific Sequence Identity with the Polynucleotides of (A), (B) or (C)
As indicated in (d), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides have a specified identity at the nucleotide level to a polynucleotide as disclosed above in paragraphs (A), (B) or (C). The percentage of identity to a reference sequence is at least 60% and, rounded upwards to the nearest integer, can be expressed as an integer selected from the group of integers consisting of from 60 to 99. Thus, for example, the percentage of identity to a reference sequence can be at least 70%, 75%, 80%, 85%, 90% or 95%.
Optionally, the polynucleotides of this embodiment will share an epitope with a polypeptide encoded by the polynucleotides of (A), (B) or (C). Thus, these polynucleotides encode a first polypeptide which elicits production of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide of (A), (B) or (C). However, the first polypeptide does not bind to antisera raised against itself when the antisera has been fully immunosorbed with the first polypeptide. Hence, the polynucleotides of this embodiment can be used to generate antibodies for use in, for example, the screening of expression libraries for nucleic acids comprising polynucleotides of (A), (B) or (C), or for purification of, or in immunoassays for, polypeptides encoded by the polynucleotides of (A), (B) or (C). The polynucleotides of this embodiment embrace nucleic acid sequences which can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.
Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 15 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT Patent Application Publication Nos. 91/17271, 91/18980, 91/19818 and 93/08278. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent Application Publication Nos. 92/05258, 92/14843 and 96/19256. See also, U.S. Pat. Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, Calif.).
E. Polynucleotides Encoding a Protein Having a Subsequence from a Prototype Polypeptide and is Cross-Reactive to the Prototype Polypeptide
As indicated in (e), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides encode a protein having a subsequence of contiguous amino acids from a prototype polypeptide of the present invention such as are provided in (a), above. The length of contiguous amino acids from the prototype polypeptide is selected from the group of integers consisting of from at least 10 to the number of amino acids within the prototype sequence. Thus, for example, the polynucleotide can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45 or 50, contiguous amino acids from the prototype polypeptide. Further, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides.
The proteins encoded by polynucleotides of this embodiment, when presented as an immunogen, elicit the production of polyclonal antibodies which specifically bind to a prototype polypeptide such as but not limited to, a polypeptide encoded by the polynucleotide of (a) or (b), above. Generally, however, a protein encoded by a polynucleotide of this embodiment does not bind to antisera raised against the prototype polypeptide when the antisera has been fully immunosorbed with the prototype polypeptide. Methods of making and assaying for antibody binding specificity/affinity are well known in the art. Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays and the like.
In a preferred assay method, fully immunosorbed and pooled antisera which is elicited to the prototype polypeptide can be used in a competitive binding assay to test the protein. The concentration of the prototype polypeptide required to inhibit 50% of the binding of the antisera to the prototype polypeptide is determined. If the amount of the protein required to inhibit binding is less than twice the amount of the prototype protein, then the protein is said to specifically bind to the antisera elicited to the immunogen. Accordingly, the proteins of the present invention embrace allelic variants, conservatively modified variants, and minor recombinant modifications to a prototype polypeptide.
A polynucleotide of the present invention optionally encodes a protein having a molecular weight as the non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptides of the present invention. Molecular weight can be readily determined by SDS-PAGE under reducing conditions. Preferably, the molecular weight is within 15% of a full length polypeptide of the present invention, more preferably within 10% or 5%, and most preferably within 3%, 2% or 1% of a full length polypeptide of the present invention. Molecular weight determination of a protein can be conveniently performed by SDS-PAGE under denaturing conditions.
Optionally, the polynucleotides of this embodiment will encode a protein having a specific activity at least 50%, 60%, 80% or 90% of the native, endogenous (i.e., non-isolated), full-length polypeptide of the present invention. Further, the proteins encoded by polynucleotides of this embodiment will optionally have a substantially similar affinity constant (K.sub.m) and/or catalytic activity (i.e., the microscopic rate constant, k.sub.cat) as the native endogenous, full-length protein. Those of skill in the art will recognize that k.sub.cat/K.sub.m value determines the specificity for competing substrates and is often referred to as the specificity constant. Proteins of this embodiment can have a k.sub.cat/K.sub.m value at least 10% of a non-isolated full-length polypeptide of the present invention as determined using the endogenous substrate of that polypeptide. Optionally, the k.sub.cat/K.sub.m value will be at least 20%, 30%, 40%, 50% and most preferably at least 60%, 70%, 80%, 90% or 95% the k.sub.cat/K.sub.m value of the non-isolated, full-length polypeptide of the present invention. Determination of k.sub.cat, K.sub.m, and k.sub.cat/K.sub.m can be determined by any number of means well known to those of skill in the art. For example, the initial rates (i.e., the first 5% or less of the reaction) can be determined using rapid mixing and sampling techniques (e.g., continuous-flow, stopped-flow, or rapid quenching techniques), flash photolysis, or relaxation methods (e.g., temperature jumps) in conjunction with such exemplary methods of measuring as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance, or radioactive procedures. Kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee plot.
F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)
As indicated in (f), above, the present invention provides isolated nucleic acids comprising polynucleotides complementary to the polynucleotides of paragraphs A-E, above. As those of skill in the art will recognize, complementary sequences base-pair throughout the entirety of their length with the polynucleotides of (A)-(E) (i.e., have 100% sequence identity over their entire length). Complementary bases associate through hydrogen bonding in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
G. Polynucleotides which are Subsequences of the Polynucleotides of (A)-(F)
As indicated in (g), above, the present invention provides isolated nucleic acids comprising polynucleotides which comprise at least 15 contiguous bases from the polynucleotides of (A) through (F) as discussed above. The length of the polynucleotide is given as an integer selected from the group consisting of from at least 15 to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of. Thus, for example, polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75 or 100 contiguous nucleotides in length from the polynucleotides of (A)-(F). Optionally, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides.
The subsequences of the present invention can comprise structural characteristics of the sequence from which it is derived. Alternatively, the subsequences can lack certain structural characteristics of the larger sequence from which it is derived. For example, a subsequence from a polynucleotide encoding a polypeptide having at least one linear epitope in common with a prototype polypeptide sequence as provided in (a), above, may encode an epitope in common with the prototype sequence. Alternatively, the subsequence may not encode an epitope in common with the prototype sequence but can be used to isolate the larger sequence by, for example, nucleic acid hybridization with the sequence from which it's derived. Subsequences can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.
Construction of Nucleic Acids
The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a monocot. In preferred embodiments the monocot is Zea mays.
The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. A polynucleotide of the present invention can be attached to a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb. Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
A. Recombinant Methods for Constructing Nucleic Acids
The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. While isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art, the following highlights some of the methods employed.
A1 mRNA Isolation and Purification
Total RNA from plant cells comprises such nucleic acids as mitochondrial RNA, chloroplastic RNA, rRNA, tRNA, hnRNA and mRNA. Total RNA preparation typically involves lysis of cells and removal of proteins, followed by precipitation of nucleic acids. Extraction of total RNA from plant cells can be accomplished by a variety of means. Frequently, extraction buffers include a strong detergent such as SDS and an organic denaturant such as guanidinium isothiocyanate, guanidine hydrochloride or phenol. Following total RNA isolation, poly(A).sup.+ mRNA is typically purified from the remainder RNA using oligo(dT) cellulose. Exemplary total RNA and mRNA isolation protocols are described in Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Total RNA and mRNA isolation kits are commercially available from vendors such as Stratagene (La Jolla, Calif.), Clonetech (Palo Alto, Calif.), Pharmacia (Piscataway, N.J.), and 5'-3' (Paoli, Pa.). See also, U.S. Pat. Nos. 5,614,391 and 5,459,253. The mRNA can be fractionated into populations with size ranges of about 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 kb. The cDNA synthesized for each of these fractions can be size selected to the same size range as its mRNA prior to vector insertion. This method helps eliminate truncated cDNA formed by incompletely reverse transcribed mRNA.
A2. Construction of a cDNA Library
Construction of a cDNA library generally entails five steps. First, first strand cDNA synthesis is initiated from a poly(A).sup.+ mRNA template using a poly(dT) primer or random hexanucleotides. Second, the resultant RNA-DNA hybrid is converted into double stranded cDNA, typically by a combination of RNAse H and DNA polymerase I (or Klenow fragment). Third, the termini of the double stranded cDNA are ligated to adaptors. Ligation of the adaptors will produce cohesive ends for cloning. Fourth, size selection of the double stranded cDNA eliminates excess adaptors and primer fragments, and eliminates partial cDNA molecules due to degradation of mRNAs or the failure of reverse transcriptase to synthesize complete first strands. Fifth, the cDNAs are ligated into cloning vectors and packaged. cDNA synthesis protocols are well known to the skilled artisan and are described in such standard references as: Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). cDNA synthesis kits are available from a variety of commercial vendors such as Stratagene or Pharmacia.
A number of cDNA synthesis protocols have been described which provide substantially pure full-length cDNA libraries. Substantially pure full-length cDNA libraries are constructed to comprise at least 90%, and more preferably at least 93% or 95% full-length inserts amongst clones containing inserts. The length of insert in such libraries can be from 0 to 8, 9, 10, 11, 12, 13 or more kilobase pairs. Vectors to accommodate inserts of these sizes are known in the art and available commercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12 kb cloning capacity).
An exemplary method of constructing a greater than 95% pure full-length cDNA library is described by Carninci, et al., (1996) Genomics, 37:327-336. In that protocol, the cap-structure of eukaryotic mRNA is chemically labeled with biotin. By using streptavidin-coated magnetic beads, only the full-length first-strand cDNA/mRNA hybrids are selectively recovered after RNase I treatment. The method provides a high yield library with an unbiased representation of the starting mRNA population. Other methods for producing full-length libraries are known in the art. See, e.g., Edery, et al., (1995) Mol. Cell. Biol., 15(6):3363-3371; and PCT Application WO 96/34981.
A3. Normalized or Subtracted cDNA Libraries
A non-normalized cDNA library represents the mRNA population of the tissue it was made from. Since unique clones are out-numbered by clones derived from highly expressed genes their isolation can be laborious. Normalization of a cDNA library is the process of creating a library in which each clone is more equally represented.
A number of approaches to normalize cDNA libraries are known in the art. One approach is based on hybridization to genomic DNA. The frequency of each hybridized cDNA in the resulting normalized library would be proportional to that of each corresponding gene in the genomic DNA. Another approach is based on kinetics. If cDNA reannealing follows second-order kinetics, rarer species anneal less rapidly and the remaining single-stranded fraction of cDNA becomes progressively more normalized during the course of the hybridization. Specific loss of any species of cDNA, regardless of its abundance, does not occur at any Cot value. Construction of normalized libraries is described in Ko, (1990) Nucl. Acids. Res., 18(19):5705-5711; Patanjali, et al., (1991) Proc. Natl. Acad. U.S.A. 88:1943-1947; U.S. Pat. Nos. 5,482,685 and 5,637,685. In an exemplary method described by Soares, et al., normalization resulted in reduction of the abundance of clones from a range of four orders of magnitude to a narrow range of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).
Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. In this procedure, cDNA prepared from one pool of mRNA is depleted of sequences present in a second pool of mRNA by hybridization. The cDNA:mRNA hybrids are removed and the remaining un-hybridized cDNA pool is enriched for sequences unique to that pool. See, Foote, et al. in, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991) Technique 3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res., 16(22):10937; Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); and, Swaroop, (1991) et al., Nucl. Acids Res., 19(8):1954. cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech).
A4. Construction of a Genomic Library
To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate molecular biological techniques and instructions sufficient to direct persons of skill through many construction, cloning, and screening methodologies are found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits for construction of genomic libraries are also commercially available.
A5. Nucleic Acid Screening and Isolation Methods
The cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.
The nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods and Applications, Innis, et al., Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.
PCR-based screening methods have also been described. Wilfinger, et al., describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques, 22(3):481-486 (1997). In that method, a primer pair is synthesized with one primer annealing to the 5' end of the sense strand of the desired cDNA and the other primer to the vector. Clones are pooled to allow large-scale screening. By this procedure, the longest possible clone is identified amongst candidate clones. Further, the PCR product is used solely as a diagnostic for the presence of the desired cDNA and does not utilize the PCR product itself. Such methods are particularly effective in combination with a full-length cDNA construction methodology, above.
B. Synthetic Methods for Constructing Nucleic Acids
The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra. Lett. 22:1859-1862; the solid phase phosphoramidite triester method described by Beaucage and Caruthers, (1981) Tetra. Letts. 22(20):1859-1862, e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter, et al., (1984) Nucleic Acids Res., 12:6159-6168; and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Recombinant Expression Cassettes
The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
A plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, the actin promoter, the F3.7 promoter, and other transcription initiation regions from various plant genes known to those of skill.
Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as "inducible" promoters. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light.
Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue. Thus, in some embodiments, the nucleic acid construct will comprise a promoter functional in a plant cell, such as in Zea mays, operably linked to a polynucleotide of the present invention. Promoters useful in these embodiments include the endogenous promoters driving expression of a polypeptide of the present invention.
In some embodiments, isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868), or isolated promoters can be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. Gene expression can be modulated under conditions suitable for plant growth so as to alter the total concentration and/or alter the composition of the polypeptides of the present invention in plant cell. Thus, the present invention provides compositions, and methods for making, heterologous promoters and/or enhancers operably linked to a native, endogenous (i.e., non-heterologous) form of a polynucleotide of the present invention.
Methods for identifying promoters with a particular expression pattern, in terms of, e.g., tissue type, cell type, stage of development, and/or environmental conditions, are well known in the art. See, e.g., The Maize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3.sup.rd edition, Chapter 6, Sprague and Dudley, Eds., American Society of Agronomy, Madison, Wis. (1988). A typical step in promoter isolation methods is identification of gene products that are expressed with some degree of specificity in the target tissue. Amongst the range of methodologies are: differential hybridization to cDNA libraries; subtractive hybridization; differential display; differential 2-D protein gel electrophoresis; DNA probe arrays; and isolation of proteins known to be expressed with some specificity in the target tissue. Such methods are well known to those of skill in the art. Commercially available products for identifying promoters are known in the art such as Clontech's (Palo Alto, Calif.) Universal GenomeWalker Kit.
For the protein-based methods, it is helpful to obtain the amino acid sequence for at least a portion of the identified protein, and then to use the protein sequence as the basis for preparing a nucleic acid that can be used as a probe to identify either genomic DNA directly, or preferably, to identify a cDNA clone from a library prepared from the target tissue. Once such a cDNA clone has been identified, that sequence can be used to identify the sequence at the 5' end of the transcript of the indicated gene. For differential hybridization, subtractive hybridization and differential display, the nucleic acid sequence identified as enriched in the target tissue is used to identify the sequence at the 5' end of the transcript of the indicated gene. Once such sequences are identified, starting either from protein sequences or nucleic acid sequences, any of these sequences identified as being from the gene transcript can be used to screen a genomic library prepared from the target organism. Methods for identifying and confirming the transcriptional start site are well known in the art.
In the process of isolating promoters expressed under particular environmental conditions or stresses, or in specific tissues, or at particular developmental stages, a number of genes are identified that are expressed under the desired circumstances, in the desired tissue, or at the desired stage. Further analysis will reveal expression of each particular gene in one or more other tissues of the plant. One can identify a promoter with activity in the desired tissue or condition but that do not have activity in any other common tissue.
To identify the promoter sequence, the 5' portions of the clones described here are analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10 bp located approximately 20 to 40 base pairs upstream of the transcription start site. Identification of the TATA box is well known in the art. For example, one way to predict the location of this element is to identify the transcription start site using standard RNA-mapping techniques such as primer extension, S1 analysis, and/or RNase protection. To confirm the presence of the AT-rich sequence, a structure-function analysis can be performed involving mutagenesis of the putative region and quantification of the mutation's effect on expression of a linked downstream reporter gene. See, e.g., The Maize Handbook, Chapter 114, Freeling and Walbot, Eds., Springer, New York, (1994).
In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element (i.e., the CAAT box) with a series of adenines surrounding the trinucleotide G (or T) N G. Messing, et al., in Genetic Engineering in Plants, Kosage, Meredith and Hollaender, Eds., pp. 221-227 (1983). In maize, there is no well conserved CAAT box but there are several short, conserved protein-binding motifs upstream of the TATA box. These include motifs for the trans-acting transcription factors involved in light regulation, anaerobic induction, hormonal regulation, or anthocyanin biosynthesis, as appropriate for each gene.
Once promoter and/or gene sequences are known, a region of suitable size is selected from the genomic DNA that is 5' to the transcriptional start, or the translational start site, and such sequences are then linked to a coding sequence. If the transcriptional start site is used as the point of fusion, any of a number of possible 5' untranslated regions can be used in between the transcriptional start site and the partial coding sequence. If the translational start site at the 3' end of the specific promoter is used, then it is linked directly to the methionine start codon of a coding sequence.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, (1988) Mol. Cell. Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev. 1:1183-1200. Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).
The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.
Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al., (1987) Meth. In Enzymol. 153:253-277. These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al., (1987) Gene 61:1-11 and Berger, et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:8402-8406. Another useful vector herein is plasmid pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, Calif.).
A polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable plant characteristics. Antisense technology can be conveniently used to gene expression in plants. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy, et al., (1988) Proc. Nat'l. Acad. Sci. (USA) 85:8805-8809; and Hiatt, et al., U.S. Pat. No. 4,801,340.
Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli, et al., (1990) The Plant Cell 2:279-289 and U.S. Pat. No. 5,034,323.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff, et al., (1988) Nature 334:585-591.
A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids Res 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J Am Chem Soc 109:1241-1243 (1987)). Meyer, et al., (1989) J Am Chem Soc 111:8517-8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, et al., (1988) Biochemistry 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home, et al., (1990) J Am Chem Soc 112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, (1986) J Am Chem Soc 108:2764-2765; Nucleic Acids Res 14:7661-7674 (1986); Feteritz, et al., (1991) J. Am. Chem. Soc. 113:4000. Various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648 and 5,681941.
Proteins
The isolated proteins of the present invention comprise a polypeptide having at least 10 amino acids encoded by any one of the polynucleotides of the present invention as discussed more fully, above, or polypeptides which are conservatively modified variants thereof. The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the present invention. Optionally, this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35 or 40 amino acids in length, often at least 50, 60, 70, 80 or 90 amino acids in length. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4 or 5.
As those of skill will appreciate, the present invention includes catalytically active polypeptides of the present invention (i.e., enzymes). Catalytically active polypeptides have a specific activity of at least 20%, 30%, or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (k.sub.cat/K.sub.m) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide. Typically, the K.sub.m will be at least 30%, 40% or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80% or 90%. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity (k.sub.cat/K.sub.m), are well known to those of skill in the art.
Generally, the proteins of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention. Further, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those of skill in the art. A preferred immunoassay is a competitive immunoassay as discussed, infra. Thus, the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.
Expression of Proteins in Host Cells
Using the nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. One of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located purification sequences. Restriction sites or termination codons can also be introduced.
A. Expression in Prokaryotes
Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake, et al., (1981) Nature 292:128). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-235; Mosbach, et al., (1983) Nature 302:543-545).
B. Expression in Eukaryotes
A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a of the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
Synthesis of heterologous proteins in yeast is well known. Sherman, et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast. Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
The sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).
Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See, Schneider, (1987) Embryol. Exp. Morphol. 27:353-365.
As with yeast, when higher animal or plant host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., (1983) J. Virol. 45:773-781). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M., Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington, Va. pp. 213-238 (1985).
Transfection/Transformation of Cells
The method of transformation/transfection is not critical to the instant invention; various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for efficient transformation/transfection may be employed.
A. Plant Transformation
A DNA sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full length protein, will be used to construct a recombinant expression cassette which can be introduced into the desired plant.
Isolated nucleic acid acids of the present invention can be introduced into plants according techniques known in the art. Generally, recombinant expression cassettes as described above and suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising, et al., (1988) Ann. Rev. Genet. 22:421-477. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment, silicon fiber delivery, or microinjection of plant cell protoplasts or embryogenic callus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, pp. 197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods, (eds. O. L. Gamborg and G. C. Phillips, Springer-Verlag Berlin Heidelberg New York, 1995). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616.
The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski, et al., (1984) Embo J. 3:2717-2722. Electroporation techniques are described in Fromm, et al., (1985) Proc. Natl. Acad. Sci. 82:5824. Ballistic transformation techniques are described in Klein, et al., (1987) Nature 327:70-73.
Agrobacterium tumefaciens-meditated transformation techniques are well described in the scientific literature. See, for example, Horsch, et al., (1984) Science 233:496-498, and Fraley, et al., (1983) Proc. Natl. Acad. Sci. 80:4803. Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium transformation of maize is described in U.S. Pat. No. 5,550,318.
Other methods of transfection or transformation include (1) Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J, In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), PCT Patent Application Number PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see, e.g., Kindle, (1990) Proc. Natl. Acad. Sci., USA 87:1228).
DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou, et al., (1983) Methods in Enzymology 101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988) Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena, et al., (1987) Nature 325:274. DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus, et al., (1987) Theor. Appl. Genet. 75:30; and Benbrook, et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells
Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art. Kuchler, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).
Synthesis of Proteins
The proteins of the present invention can be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A; Merrifield, et al., (1963) J. Am. Chem. Soc. 85:2149-2156, and Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide)) is known to those of skill.
Purification of Proteins
The proteins of the present invention may be purified by standard techniques well known to those of skill in the art. Recombinantly produced proteins of the present invention can be directly expressed or expressed as a fusion protein. The recombinant protein is purified by a combination of cell lysis (e.g., sonication, French press) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein.
The proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Pat. No. 4,511,503. The protein may then be isolated from cells expressing the protein and further purified by standard protein chemistry techniques as described herein. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.
Transgenic Plant Regeneration
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with a polynucleotide of the present invention. For transformation and regeneration of maize see, Gordon-Kamm, et al., (1990) The Plant Cell 2:603-618.
Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans, et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
The regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch, et al., (1985) Science 227:1229-1231. In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley, et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 80:4803. This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.
Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee, et al., (1987) Ann. Rev. of Plant Phys. 38:467-486. The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, Weissbach and Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3.sup.rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988).
One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.
Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.
Modulating Polypeptide Levels and/or Composition
The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or composition of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e., the ratio of the polypeptides of the present invention) in a plant. The method comprises transforming a plant cell with a recombinant expression cassette comprising a polynucleotide of the present invention as described above to obtain a transformed plant cell, growing the transformed plant cell under plant forming conditions, and inducing expression of a polynucleotide of the present invention in the plant for a time sufficient to modulate concentration and/or composition in the plant or plant part.
In some embodiments, the content and/or composition of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated gene of the present invention to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868. And in some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or composition of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and discussed briefly, above.
In general, concentration or composition is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a native control plant, plant part, or cell lacking the aforementioned recombinant expression cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, above. Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound. Inducible promoters and inducing compounds which activate expression from these promoters are well known in the art. In preferred embodiments, the polypeptides of the present invention are modulated in monocots, particularly maize.
Molecular Markers
The present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention. Preferably, the plant is a monocot, such as maize or sorghum. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997). For molecular marker methods, see generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R. G. Landis Company, Austin, Tex., pp. 7-21.
The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs). RFLPs are the product of allelic differences between DNA restriction fragments caused by nucleotide sequence variability. As is well known to those of skill in the art, RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP. Thus, the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2 or 1 cM of a gene of the present invention.
In the present invention, the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention. In preferred embodiments, the probes are selected from polynucleotides of the present invention. Typically, these probes are cDNA probes or Pst I genomic clones. The length of the probes is discussed in greater detail, above, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40 or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length. Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement. Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and SstI. As used herein the term "restriction enzyme" includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.
The method of detecting an RFLP comprises the steps of (a) digesting genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; (c) detecting therefrom a RFLP. Other methods of differentiating polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCP); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CMC). Exemplary polymorphic variants are provided in Table I, above. Thus, the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe. Generally, the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA). The nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the nucleic acid probe comprises a polynucleotide of the present invention.
UTR's and Codon Preference
In general, translational efficiency has been found to be regulated by specific sequence elements in the 5' non-coding or untranslated region (5' UTR) of the RNA. Positive sequence motifs include translational initiation consensus sequences (Kozak, (1987) Nucleic Acids Res. 15:8125) and the 7-methylguanosine cap structure (Drummond, et al., (1985) Nucleic Acids Res. 13:7375). Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, above, Rao, et al., (1988) Mol. and Cell. Biol. 8:284). Accordingly, the present invention provides 5' and/or 3' UTR regions for modulation of translation of heterologous coding sequences.
Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host or to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference" available from the University of Wisconsin Genetics Computer Group (see, Devereaux, et al., (1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides that can be used to determine a codon usage frequency can be any integer from 1 to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
Sequence Shuffling
The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT Patent Application Publication Number 96/19256. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509. Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be a decreased K.sub.m and/or increased K.sub.cat over the wild-type protein as provided herein. In other embodiments, a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.
Generic and Consensus Sequences
Polynucleotides and polypeptides of the present invention further include those having: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and, (b) a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention. The generic sequence of the present invention comprises each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide, sequence, respectively. The individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phyla, or kingdoms. For example, a polynucleotide having a consensus sequences from a gene family of Zea mays can be used to generate antibody or nucleic acid probes or primers to other Gramineae species such as wheat, rice, or sorghum. Alternatively, a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence will be at least 9, 10, 15, 20, 25, 30 or 40 amino acids in length, or 20, 30, 40, 50, 100 or 150 nucleotides in length. As those of skill in the art are aware, a conservative amino acid substitution can be used for amino acids which differ amongst aligned sequence but are from the same conservative substitution group as discussed above. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.
Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous, or paralogous sequences as provided herein. Optionally, similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability (P(N)). Various suppliers of sequence-analysis software are listed in chapter 7 of Current Protocols in Molecular Biology, F. M. Ausubel, et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 30). A polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001, and most preferably less than about 0.0001, or 0.00001. Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, Wis.) PILEUP software, Vector NTI's (North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.
Detection of Nucleic Acids
The present invention further provides methods for detecting a polynucleotide of the present invention in a nucleic acid sample suspected of comprising a polynucleotide of the present invention, such as a plant cell lysate, particularly a lysate of corn. In some embodiments, a gene of the present invention or portion thereof can be amplified prior to the step of contacting the nucleic acid sample with a polynucleotide of the present invention. The nucleic acid sample is contacted with the polynucleotide to form a hybridization complex. The polynucleotide hybridizes under stringent conditions to a gene encoding a polypeptide of the present invention. Formation of the hybridization complex is used to detect a gene encoding a polypeptide of the present invention in the nucleic acid sample. Those of skill will appreciate that an isolated nucleic acid comprising a polynucleotide of the present invention should lack cross-hybridizing sequences in common with non-target genes that would yield a false positive result.
Detection of the hybridization complex can be achieved using any number of well known methods. For example, the nucleic acid sample, or a portion thereof, may be assayed by hybridization formats including but not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays. Briefly, in solution (or liquid) phase hybridizations, both the target nucleic acid and the probe or primer are free to interact in the reaction mixture. In solid phase hybridization assays, probes or primers are typically linked to a solid support where they are available for hybridization with target nucleic in solution. In mixed phase, nucleic acid intermediates in solution hybridize to target nucleic acids in solution as well as to a nucleic acid linked to a solid support. In in situ hybridization, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis. The following articles provide an overview of the various hybridization assay formats: Singer, et al., (1986) Biotechniques 4(3):230-250; Haze, et al., (1984) Methods in Virology, Vol. VII, pp. 189-226; Wilkinson, The theory and practice of in situ hybridization in: In situ Hybridization, D. G. Wilkinson, Ed., IRL Press, Oxford University Press, Oxford; and Nucleic Acid Hybridization: A Practical Approach, Hames, B. D. and Higgins, S. J., Eds., IRL Press (1987).
Nucleic Acid Labels and Detection Methods
The means by which nucleic acids of the present invention are labeled is not a critical aspect of the present invention and can be accomplished by any number of methods currently known or later developed. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
Nucleic acids of the present invention can be labeled by any one of several methods typically used to detect the presence of hybridized nucleic acids. One common method of detection is the use of autoradiography using probes labeled with .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P, or the like. The choice of radio-active isotope depends on research preferences due to ease of synthesis, stability, and half lives of the selected isotopes. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents or enzymes. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation. Labeling the nucleic acids of the present invention is readily achieved such as by the use of labeled PCR primers.
In some embodiments, the label is simultaneously incorporated during the amplification step in the preparation of the nucleic acids. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In another embodiment, transcription amplification using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.
Non-radioactive probes are often labeled by indirect means. For example, a ligand molecule is covalently bound to the probe. The ligand then binds to an anti-ligand molecule which is either inherently detectable or covalently bound to a detectable signal system, such as an enzyme, a fluorophore, or a chemiluminescent compound. Enzymes of interest as labels will primarily be hydrolases, such as phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. Ligands and anti-ligands may be varied widely. Where a ligand has a natural anti-ligand, namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
Probes can also be labeled by direct conjugation with a label. For example, cloned DNA probes have been coupled directly to horseradish peroxidase or alkaline phosphatase, (Renz and Kurz, (1984) A Colorimetric Method for DNA Hybridization, Nucl. Acids Res. 12:3435-3444) and synthetic oligonucleotides have been coupled directly with alkaline phosphatase (Jablonski, et al., (1986) Preparation of Oligodeoxynucleotide-Alkaline Phosphatase Conjugates and Their Use as Hybridization Probes, Nuc. Acids. Res. 14:6115-6128; and Li, et al., (1987) Enzyme-linked Synthetic Oligonucleotide probes: Non-Radioactive Detection of Enterotoxigenic Escherichia Coli in Faeca Specimens, Nucl. Acids Res. 15:5275-5287).
Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
Antibodies to Proteins
Antibodies can be raised to a protein of the present invention, including individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill. The following discussion is presented as a general overview of the techniques available; however, one of skill will recognize that many variations upon the following methods are known.
A number of immunogens are used to produce antibodies specifically reactive with a protein of the present invention. An isolated recombinant, synthetic, or native polynucleotide of the present invention are the preferred immunogens (antigen) for the production of monoclonal or polyclonal antibodies. Those of skill will readily understand that the proteins of the present invention are typically denatured, and optionally reduced, prior to formation of antibodies for screening expression libraries or other assays in which a putative protein of the present invention is expressed or denatured in a non-native secondary, tertiary, or quartenary structure. Non-isolated polypeptides of the present invention can be used either in pure or impure form.
The protein of the present invention is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the protein of the present invention. Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen (antigen), preferably a purified protein, a protein coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a protein incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the protein of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein is performed where desired (See, e.g., Coligan, Current Protocols in Immunology, Wiley/Greene, NY (1991); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY (1989)).
Antibodies, including binding fragments and single chain recombinant versions thereof, against predetermined fragments of a protein of the present invention are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above. Typically, the immunogen of interest is a protein of at least about 5 amino acids, more typically the protein is 10 amino acids in length, preferably, 15 amino acids in length and more preferably the protein is 20 amino acids in length or greater. The peptides are typically coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in an immunization vector. Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.
Monoclonal antibodies are prepared from cells secreting the desired antibody. Monoclonals antibodies are screened for binding to a protein from which the immunogen was derived. Specific monoclonal and polyclonal antibodies will usually have an antibody binding site with an affinity constant for its cognate monovalent antigen at least between 10.sup.6-10.sup.7, usually at least 10.sup.8, preferably at least 10.sup.9, more preferably at least 10.sup.10, and most preferably at least 10.sup.11 liters/mole.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Basic and Clinical Immunology, 4th ed., Stites, et al., Eds., Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986); and Kohler and Milstein, (1975) Nature 256:495-497. Summarized briefly, this method proceeds by injecting an animal with an immunogen comprising a protein of the present invention. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse, et al., (1989) Science 246:1275-1281; and Ward, et al., (1989) Nature 341:544-546; and Vaughan, et al., (1996) Nature Biotechnology, 14:309-314). Alternatively, high avidity human monoclonal antibodies can be obtained from transgenic mice comprising fragments of the unrearranged human heavy and light chain Ig loci (i.e., minilocus transgenic mice). Fishwild, et al., (1996) Nature Biotech. 14:845-851. Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al., (1989) Proc. Natl. Acad. Sci. 86:10029-10033.
The antibodies of this invention are also used for affinity chromatography in isolating proteins of the present invention. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified protein are released.
The antibodies can be used to screen expression libraries for particular expression products such as normal or abnormal protein. Usually the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
Antibodies raised against a protein of the present invention can also be used to raise anti-idiotypic antibodies. These are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.
Frequently, the proteins and antibodies of the present invention will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
Protein Immunoassays
Means of detecting the proteins of the present invention are not critical aspects of the present invention. In a preferred embodiment, the proteins are detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288 and 4,837,168). For a review of the general immunoassays, see also, Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites and Terr, Eds. (1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980); Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam (1985); Harlow and Lane, above; Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles and Practice of Immunoassaysm, Price and Newman Eds., Stockton Press, NY (1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY (1988). Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case, a protein of the present invention). The capture agent is a moiety that specifically binds to the analyte. In a preferred embodiment, the capture agent is an antibody that specifically binds a protein(s) of the present invention. The antibody may be produced by any of a number of means known to those of skill in the art as described herein.
Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled protein of the present invention or a labeled antibody specifically reactive to a protein of the present invention. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
In a preferred embodiment, the labeling agent is a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval, et al., (1973) J. Immunol. 111:1401-1406 and Akerstrom, et al., (1985) J. Immunol. 135:2589-2542).
Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10.degree. C. to 40.degree. C.
While the details of the immunoassays of the present invention may vary with the particular format employed, the method of detecting a protein of the present invention in a biological sample generally comprises the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to a protein of the present invention. The antibody is allowed to bind to the protein under immunologically reactive conditions, and the presence of the bound antibody is detected directly or indirectly.
A Non-Competitive Assay Formats
Immunoassays for detecting proteins of the present invention include competitive and noncompetitive formats. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e., a protein of the present invention) is directly measured. In one preferred "sandwich" assay, for example, the capture agent (e.g., an antibody specifically reactive, under immunoreactive conditions, to a protein of the present invention) can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the protein present in the test sample. The protein thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
B. Competitive Assay Formats
In competitive assays, the amount of analyte present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (e.g., a protein of the present invention) displaced (or competed away) from a capture agent (e.g., an antibody specifically reactive, under immunoreactive conditions, to the protein) by the analyte present in the sample. In one competitive assay, a known amount of analyte is added to the sample and the sample is then contacted with a capture agent that specifically binds a protein of the present invention. The amount of protein bound to the capture agent is inversely proportional to the concentration of analyte present in the sample.
In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of protein bound to the antibody may be determined either by measuring the amount of protein present in a protein/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein. The amount of protein may be detected by providing a labeled protein.
A hapten inhibition assay is another preferred competitive assay. In this assay a known analyte, (such as a protein of the present invention) is immobilized on a solid substrate. A known amount of antibody specifically reactive, under immunoreactive conditions, to the protein is added to the sample, and the sample is then contacted with the immobilized protein. In this case, the amount of antibody bound to the immobilized protein is inversely proportional to the amount of protein present in the sample. Again, the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
C. Generation of Pooled Antisera for Use in Immunoassays
A protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen is determined in an immunoassay. The immunoassay uses a polyclonal antiserum which is raised to a polypeptide of the present invention (i.e., the immunogenic polypeptide). This antiserum is selected to have low crossreactivity against other proteins and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay (e.g., by immunosorbtion of the antisera with a protein of different substrate specificity (e.g., a different enzyme) and/or a protein with the same substrate specificity but of a different form).
In order to produce antisera for use in an immunoassay, a polypeptide of the present invention is isolated as described herein. For example, recombinant protein can be produced in a mammalian or other eukaryotic cell line. An inbred strain of mice is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, Harlow and Lane, above). Alternatively, a synthetic polypeptide derived from the sequences disclosed herein and conjugated to a carrier protein is used as an immunogen. Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10.sup.4 or greater are selected and tested for their cross reactivity against polypeptides of different forms or substrate specificity, using a competitive binding immunoassay such as the one described in Harlow and Lane, above, at pages 570-573. Preferably, two or more distinct forms of polypeptides are used in this determination. These distinct types of polypeptides are used as competitors to identify antibodies which are specifically bound by the polypeptide being assayed for. The competitive polypeptides can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein.
Immunoassays in the competitive binding format are used for crossreactivity determinations. For example, the immunogenic polypeptide is immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the immunogenic polypeptide. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with a distinct form of a polypeptide are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with a distinct form of a polypeptide.
The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described herein to compare a second "target" polypeptide to the immunogenic polypeptide. In order to make this comparison, the two polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the antisera to the immobilized protein is determined using standard techniques. If the amount of the target polypeptide required is less than twice the amount of the immunogenic polypeptide that is required, then the target polypeptide is said to specifically bind to an antibody generated to the immunogenic protein. As a final determination of specificity, the pooled antisera is fully immunosorbed with the immunogenic polypeptide until no binding to the polypeptide used in the immunosorbtion is detectable. The fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If no reactivity is observed, then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
D. Other Assay Formats
In a particularly preferred embodiment, Western blot (immunoblot) analysis is used to detect and quantify the presence of protein of the present invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind a protein of the present invention. The antibodies specifically bind to the protein on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies.
E. Quantification of Proteins.
The proteins of the present invention may be detected and quantified by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-1 inked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
F. Reduction of Non-Specific Binding
One of skill will appreciate that it is often desirable to reduce non-specific binding in immunoassays and during analyte purification. Where the assay involves an antigen, antibody, or other capture agent immobilized on a solid substrate, it is desirable to minimize the amount of non-specific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used.
G. Immunoassay Labels
The labeling agent can be, e.g., a monoclonal antibody, a polyclonal antibody, a binding protein or complex, or a polymer such as an affinity matrix, carbohydrate or lipid. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Detection may proceed by any known method, such as immunoblotting, western analysis, gel-mobility shift assays, fluorescent in situ hybridization analysis (FISH), tracking of radioactive or bioluminescent markers, nuclear magnetic resonance, electron paramagnetic resonance, stopped-flow spectroscopy, column chromatography, capillary electrophoresis, or other methods which track a molecule based upon an alteration in size and/or charge. The particular label or detectable group used in the assay is not a critical aspect of the invention. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels or colored glass or plastic beads, as discussed for nucleic acid labels, above.
The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, ease of conjugation of the compound, stability requirements, available instrumentation, and disposal provisions.
Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Pat. No. 4,391,904, which is incorporated herein by reference.
Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
Assays for Compounds that Modulate Enzymatic Activity or Expression
The present invention also provides means for identifying compounds that bind to (e.g., substrates), and/or increase or decrease (i.e., modulate) the enzymatic activity of, catalytically active polypeptides of the present invention. The method comprises contacting a polypeptide of the present invention with a compound whose ability to bind to or modulate enzyme activity is to be determined. The polypeptide employed will have at least 20%, preferably at least 30% or 40%, more preferably at least 50% or 60% and most preferably at least 70% or 80% of the specific activity of the native, full-length polypeptide of the present invention (e.g., enzyme). Generally, the polypeptide will be present in a range sufficient to determine the effect of the compound, typically about 1 nM to 10 .mu.M. Likewise, the compound will be present in a concentration of from about 1 nM to 10 .mu.M. Those of skill will understand that such factors as enzyme concentration, ligand concentrations (i.e., substrates, products, inhibitors, activators), pH, ionic strength, and temperature will be controlled so as to obtain useful kinetic data and determine the presence of absence of a compound that binds or modulates polypeptide activity. Methods of measuring enzyme kinetics is well known in the art. See, e.g., Segel, Biochemical Calculations, 2.sup.nd ed., John Wiley and Sons, New York (1976).
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
EXAMPLE 1
This example describes the construction cDNA libraries.
Total RNA Isolation
Total RNA was isolated from corn tissues with TRizol Reagent (Life Technology Inc. Gaithersburg, Md.) using a modification of the guanidine isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi (Chomczynski and Sacchi, (1987) Anal. Biochem. 162:156). In brief, plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIzol Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.
Poly(A)+ RNA Isolation
The selection of poly(A)+ RNA from total RNA was performed using PolyATact system (Promega Corporation. Madison, Wis.). In brief, biotinylated oligo(dT) primers were used to hybridize to the 3' poly(A) tails on mRNA. The hybrids were captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand. The mRNA was washed at high stringent condition and eluted by RNase-free deionized water.
cDNA Library Construction
cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the SuperScript Plasmid System (Life Technology Inc. Gaithersburg, Md.). The first stand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by SuperScript Reverse Transcriptase II at 45.degree. C. The second strand of cDNA was labeled with alpha-.sup.32P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA molecules smaller than 500 base pairs and unligated adapters were removed by Sephacryl-S400 chromatography. The selected cDNA molecules were ligated into pSPORT1 vector in between of Not I and Sal I sites.
EXAMPLE 2
This example describes cDNA sequencing and library subtraction.
Sequencing Template Preparation
Individual colonies were picked and DNA was prepared either by PCR with M13 forward primers and M13 reverse primers, or by plasmid isolation. All the cDNA clones were sequenced using M13 reverse primers.
Q-bot Subtraction Procedure
cDNA libraries subjected to the subtraction procedure were plated out on 22.times.22 cm.sup.2 agar plate at density of about 3,000 colonies per plate. The plates were incubated in a 37.degree. C. incubator for 12-24 hours. Colonies were picked into 384-well plates by a robot colony picker, Q-bot (GENETIX Limited). These plates were incubated overnight at 37.degree. C.
Once sufficient colonies were picked, they were pinned onto 22.times.22 cm.sup.2 nylon membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864 colonies. These membranes were placed onto agar plate with appropriate antibiotic. The plates were incubated at 37.degree. C. for overnight.
After colonies were recovered on the second day, these filters were placed on filter paper prewetted with denaturing solution for four minutes, then were incubated on top of a boiling water bath for additional four minutes. The filters were then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution was removed by placing the filters on dry filter papers for one minute, the colony side of the filters were place into Proteinase K solution, incubated at 37.degree. C. for 40-50 minutes. The filters were placed on dry filter papers to dry overnight. DNA was then cross-linked to nylon membrane by UV light treatment.
Colony hybridization was conducted as described by Sambrook, J., Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A laboratory Manual, 2.sup.nd Edition). The following probes were used in colony hybridization: 1. First strand cDNA from the same tissue as the library was made from to remove the most redundant clones. 2. 48-192 most redundant cDNA clones from the same library based on previous sequencing data. 3. 192 most redundant cDNA clones in the entire corn partial sequence database. 4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA. 5. cDNA clones derived from rRNA. The image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.
EXAMPLE 3
This example describes identification of the gene from a computer homology search.
Gene identities were determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, et al., (1993) J. Mol. Biol. 215:403-410; see also, www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm. The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States, (1993) Nature Genetics 3:266-272) provided by the NCBI. In some cases, the sequencing data from two or more clones containing overlapping segments of DNA were used to construct contiguous DNA sequences.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.
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68 DNA Zea mays CDS (63)...(3239) unsure (3487)...(3487) misc_feature (568) n = A,T,C or G cccac gcgtccggag ctcgtcgtca tccgccgcga tggcgagcca gggccgaagc 6g gac cag cgg aac ggc cag gtg tgc cag att tgc ggc gac gac Asp Gln Arg Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Asp ggg cgc aac ccc gac ggg gag cct ttc gtg gcc tgc aac gag tgc Gly Arg Asn Pro Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys 2 gcc ttc ccc atc tgc cgg gac tgc tac gag tac gag cgc cgc gag ggc 2Phe Pro Ile Cys Arg Asp Cys Tyr Glu Tyr Glu Arg Arg Glu Gly 35 4g cag aac tgc ccc cag tgc aag acc cgc ttc aag cgc ttc aag ggg 25ln Asn Cys Pro Gln Cys Lys Thr Arg Phe Lys Arg Phe Lys Gly 5 tgc gcg cgc gtg ccc ggg gac gag gag gag gac ggc gtc gac gac ctg 299 Cys Ala Arg Val Pro Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu 65 7g aac gag ttc aac tgg agc gac aag cac gac tcc cag tac ctc gcc 347 Glu Asn Glu Phe Asn Trp Ser Asp Lys His Asp Ser Gln Tyr Leu Ala 8 95 gag tcc atg ctc cac gcc cac atg agc tac ggc cgc ggc gcc gac ctc 395 Glu Ser Met Leu His Ala His Met Ser Tyr Gly Arg Gly Ala Asp Leu ggc gtg ccg cag cca ttc cac ccc atc ccc aat gtt ccc ctc ctc 443 Asp Gly Val Pro Gln Pro Phe His Pro Ile Pro Asn Val Pro Leu Leu aac gga cag atg gtc gat gac atc ccg ccg gac cag cac gcc ctt 49sn Gly Gln Met Val Asp Asp Ile Pro Pro Asp Gln His Ala Leu ccc tcg ttc gtg ggt ggc ggg ggg aag agg att cac cct ctc ccg 539 Val Pro Ser Phe Val Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro gcg gat ccc aac ctt cct gtg caa ccg agg tct atg gac cct tcc 587 Tyr Ala Asp Pro Asn Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser aag gat ctc gcc gca tat ggc tac ggg agc gta gca tgg aag gag agg 635 Lys Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg gag agc tgg aag cag aag cag gag agg atg cac cag acg agg aac 683 Met Glu Ser Trp Lys Gln Lys Gln Glu Arg Met His Gln Thr Arg Asn 2ggc ggc ggc gat gat ggt gat gat gca gat cta cca cta atg gat 73ly Gly Gly Asp Asp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp 222ct aga cag cca ttg tcc aga aag atc ccg ctt cct tca agc caa 779 Glu Ala Arg Gln Pro Leu Ser Arg Lys Ile Pro Leu Pro Ser Ser Gln 225 23tc aac ccc tat agg atg att ata ata att cgg cta gtg gtt ttg tgt 827 Ile Asn Pro Tyr Arg Met Ile Ile Ile Ile Arg Leu Val Val Leu Cys 245tc ttc ttc cac tac cga gtg atg cat ccg gtg cct gat gca ttt gct 875 Phe Phe Phe His Tyr Arg Val Met His Pro Val Pro Asp Ala Phe Ala 267gg ctc ata tct gtg atc tgt gaa att tgg ttt gcc atg tct tgg 923 Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp 275 28tt ctt gac cag ttt cca aag tgg ttt cct atc gag agg gaa acc tat 97eu Asp Gln Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu Thr Tyr 29gac cgg ctg agt tta agg ttt gac aag gaa ggg cat cct tct caa u Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly His Pro Ser Gln 33gcc cct gtt gat ttc ttt gtc agt acg gtt gat ccc ttg aag gaa u Ala Pro Val Asp Phe Phe Val Ser Thr Val Asp Pro Leu Lys Glu 323ct cca ttg gtc act gct aat act gtt cta tct atc ctt tcg gtg gat o Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ser Val Asp 345ca gtt gat aag gtt tca tgc tac gtt tct gat gat ggt gct gcc r Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala 355 36tg ctg aca ttt gaa gca ttg tct gaa aca tct gaa ttt gca aag aaa t Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys 378tt cct ttc tgc aaa aga tat agc ctt gag cct cgt gct cca gag p Val Pro Phe Cys Lys Arg Tyr Ser Leu Glu Pro Arg Ala Pro Glu 385 39gg tac ttc caa cag aag ata gac tac ctg aaa gac aag gtg gcg cca p Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Ala Pro 44aac ttt gtt aga gaa cgg aga gca atg aag aga gag tat gag gaa ttc n Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe 423tc aga atc aat gcc ttg gtt gct aaa gcc caa aag gtt cct gag s Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu 435 44aa gga tgg aca atg cag gat gga act cca tgg ccc gga aat aat gtc u Gly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Val 456at cat cct gga atg att cag gtt ttc ctt ggt caa agt ggt ggc g Asp His Pro Gly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly 465 47at gat gtg gaa gga aat gag ctg cct cga ttg gtt tat gtt tca aga s Asp Val Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg 489aa aaa cgg cca ggc tac aac cat cac aag aag gct ggt gct atg aat u Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn 55ttg gtc cga gtc tct gct gta cta act aat gct cct tat ttg ctg a Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Leu Leu 5525 aac ttg gat tgt gat cac tat atc aat aat agt aag gct ata aag gaa n Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu 534tg tgt ttt atg atg gat cct ttg ctt gga aag aaa gtt tgc tat a Met Cys Phe Met Met Asp Pro Leu Leu Gly Lys Lys Val Cys Tyr 545 55tg cag ttt cct caa aga ttt gat ggg att gat cgc cat gat cga tat l Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr 567ct aac aga aat gtt gtc ttt ttc gat atc aac atg aaa ggt ttg gat a Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp 589tc cag ggc cca att tat gtg ggt act gga tgt gtc ttc aga agg y Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys Val Phe Arg Arg 595 6cag gca tta tat ggc tac gat gct ccc aaa aca aag aag cca cca tca n Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser 662ct tgc aac tgc tgg cca aag tgg tgc att tgc tgt tgc tgt ttt g Thr Cys Asn Cys Trp Pro Lys Trp Cys Ile Cys Cys Cys Cys Phe 625 63gt aac agg aag acc aag aag aag acc aag acc tct aaa cct aaa ttt 2 Asn Arg Lys Thr Lys Lys Lys Thr Lys Thr Ser Lys Pro Lys Phe 645ag aag ata aag aaa ctt ttt aag aaa aag gaa aat caa gcc cct gca 2 Lys Ile Lys Lys Leu Phe Lys Lys Lys Glu Asn Gln Ala Pro Ala 667ct ctt ggt gaa att gat gaa gcc gct cca gga gct gaa aat gaa 2 Ala Leu Gly Glu Ile Asp Glu Ala Ala Pro Gly Ala Glu Asn Glu 675 68ag gct agt att gta aat caa cag aag ttg gaa aag aaa ttt ggc cag 2 Ala Ser Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln 69tca gtt ttt gtt gca tcc aca ctt ctt gag aat ggt gga acc ctg 22Ser Val Phe Val Ala Ser Thr Leu Leu Glu Asn Gly Gly Thr Leu 77agt gcc agt cca gct tct ctt ctg aag gaa gct ata cat gtc atc 2267 Lys Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile 723gt tgt gga tat gaa gac aaa aca ggc tgg gga aaa gat att ggt tgg 23Cys Gly Tyr Glu Asp Lys Thr Gly Trp Gly Lys Asp Ile Gly Trp 745at gga tca gtc aca gaa gat att ctt act ggg ttt aag atg cac 2363 Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His 755 76gc cat ggt tgg cgg tca att tac tgc ata cct aaa cgg gcc gcc ttc 24His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Ala Ala Phe 778gt tcc gca cct ctc aat ctt tcc gat cgt ttt cac cag gtt ctt 2459 Lys Gly Ser Ala Pro Leu Asn Leu Ser Asp Arg Phe His Gln Val Leu 785 79gg tgg gct ctt ggt tca att gaa att ttg ttc agc aac cac tgc cct 25Trp Ala Leu Gly Ser Ile Glu Ile Leu Phe Ser Asn His Cys Pro 88ctc tgg tat ggg tat ggt ggt gga cta aag ttc ctg gaa agg ttt tcg 2555 Leu Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser 823tt aac tcc atc gta tac cct tgg aca tct atc ccg ctc ttg gcc 26Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala 835 84at tgc aca ttg cct gcc atc tgc ttg ctg aca ggg aaa ttt atc acg 265ys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr 856ag ctt aac aat gtt gcc agc ctc tgg ttc atg tca ctt ttc atc 2699 Pro Glu Leu Asn Asn Val Ala Ser Leu Trp Phe Met Ser Leu Phe Ile 865 87gc att ttt gct acg agc atc ctg gaa atg aga tgg agt ggt gta ggc 2747 Cys Ile Phe Ala Thr Ser Ile Leu Glu Met Arg Trp Ser Gly Val Gly 889tc gat gac tgg tgg aga aac gag cag ttt tgg gtc att gga ggc gtg 2795 Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val 99tca cat ctc ttt gct gtg ttc cag gga ctc ctc aag gtc ata gct 2843 Ser Ser His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Ile Ala 9925 ggt gta gac acg agc ttc act gtg aca tcc aag ggc gga gac gac gag 289al Asp Thr Ser Phe Thr Val Thr Ser Lys Gly Gly Asp Asp Glu 934tc tca gag ctg tac aca ttc aaa tgg acg acc ctt ctg ata cct 2939 Glu Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro 945 95cg aca acc ctg ctc cta ctg aac ttc att gga gtg gta gct ggc atc 2987 Pro Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala Gly Ile 967cc aat gcg atc aac aac gga tat gaa tca tgg ggc ccc ctg ttc ggg 3 Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly 989tc ttc ttt gca ttt tgg gtg atc gtc cat ctt tac ccg ttc ctc 3 Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu 995 ggt ctg gtt ggg agg cag aac agg acg cca acg att gtc att gtc 3 Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val tgg tcc atc ctc ctg gct tcg atc ttc tcg ctg ctt tgg gtc cgg atc 3 Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile 3gac ccg ttc ctt gcg aag gat gat ggt ccc ctg ttg gag gag tgt ggt 3227 Asp Pro Phe Leu Ala Lys Asp Asp Gly Pro Leu Leu Glu Glu Cys Gly 45 55 ctg gat tgc aac taggaggtca gcacgtggac ttccccgtca gtgtgtggtc 3279 Leu Asp Cys Asn gaagaagtat ttttgcagat gttttgtgcc catatttctt tactcaattt ttgtccctct 3339 gtagattgaa acaaggggtg aaggggaaaa aaagtacttg tatttctttt gttccatggt 3399 ggtggtggtg gtgggcggct cagcctcgtg agtgcaatat tgggcaaacc ggaggttgcg 3459 gcaaccttgt gcagttcgtc cacgaatnta ctagggatga tcgcgaccaa tcaatcaatc 35accgag ttcaattgtt caaaaaaaaa aaaaaaaaag ggcggccgc 3568 2 T Zea mays 2 Met Asp Gln Arg Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Asp Val Arg Asn Pro Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala 2 Phe Pro Ile Cys Arg Asp Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr 35 4n Asn Cys Pro Gln Cys Lys Thr Arg Phe Lys Arg Phe Lys Gly Cys 5 Ala Arg Val Pro Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Glu 65 7 Asn Glu Phe Asn Trp Ser Asp Lys His Asp Ser Gln Tyr Leu Ala Glu 85 9r Met Leu His Ala His Met Ser Tyr Gly Arg Gly Ala Asp Leu Asp Val Pro Gln Pro Phe His Pro Ile Pro Asn Val Pro Leu Leu Thr Gly Gln Met Val Asp Asp Ile Pro Pro Asp Gln His Ala Leu Val Ser Phe Val Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Tyr Ala Asp Pro Asn Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met Ser Trp Lys Gln Lys Gln Glu Arg Met His Gln Thr Arg Asn Asp 2Gly Gly Asp Asp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu 222rg Gln Pro Leu Ser Arg Lys Ile Pro Leu Pro Ser Ser Gln Ile 225 234ro Tyr Arg Met Ile Ile Ile Ile Arg Leu Val Val Leu Cys Phe 245 25he Phe His Tyr Arg Val Met His Pro Val Pro Asp Ala Phe Ala Leu 267eu Ile Ser Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile 275 28eu Asp Gln Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu Thr Tyr Leu 29Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly His Pro Ser Gln Leu 33Ala Pro Val Asp Phe Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro 325 33ro Leu Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr 345al Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met 355 36eu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp 378ro Phe Cys Lys Arg Tyr Ser Leu Glu Pro Arg Ala Pro Glu Trp 385 39Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Ala Pro Asn 44Val Arg Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys 423rg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu 435 44ly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Val Arg 456is Pro Gly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly His 465 478al Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu 485 49ys Arg Pro Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala 55Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Leu Leu Asn 5525 Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala 534ys Phe Met Met Asp Pro Leu Leu Gly Lys Lys Val Cys Tyr Val 545 556he Pro Gln Arg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ala 565 57sn Arg Asn Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly 589ln Gly Pro Ile Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln 595 6Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg 662ys Asn Cys Trp Pro Lys Trp Cys Ile Cys Cys Cys Cys Phe Gly 625 634rg Lys Thr Lys Lys Lys Thr Lys Thr Ser Lys Pro Lys Phe Glu 645 65ys
Ile Lys Lys Leu Phe Lys Lys Lys Glu Asn Gln Ala Pro Ala Tyr 667eu Gly Glu Ile Asp Glu Ala Ala Pro Gly Ala Glu Asn Glu Lys 675 68la Ser Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser 69Val Phe Val Ala Ser Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys 77Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser 725 73ys Gly Tyr Glu Asp Lys Thr Gly Trp Gly Lys Asp Ile Gly Trp Ile 745ly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys 755 76is Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Ala Ala Phe Lys 778er Ala Pro Leu Asn Leu Ser Asp Arg Phe His Gln Val Leu Arg 785 79Ala Leu Gly Ser Ile Glu Ile Leu Phe Ser Asn His Cys Pro Leu 88Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr 823sn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala Tyr 835 84ys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr Pro 856eu Asn Asn Val Ala Ser Leu Trp Phe Met Ser Leu Phe Ile Cys 865 878he Ala Thr Ser Ile Leu Glu Met Arg Trp Ser Gly Val Gly Ile 885 89sp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser 99His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Ile Ala Gly 9925 Val Asp Thr Ser Phe Thr Val Thr Ser Lys Gly Gly Asp Asp Glu Glu 934er Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro 945 956hr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala Gly Ile Ser 965 97sn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys 989he Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys 995 Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp 3o Phe Leu Ala Lys Asp Asp Gly Pro Leu Leu Glu Glu Cys Gly Leu 5Asp Cys Asn 3 25 DNA Zea mays 3 atggaccagc ggaacggcca ggtgt 25 4 25 DNA Zea mays 4 ctagttgcaa tccagaccac actcc 25 5 3773 DNA Zea mays CDS (338)...(3568) 5 gtcgacccac gcgtccgcta ggatcaaaac cgtctcgccg ctgcaataat cttttgtcaa 6aatcc ctcgcgtcga cagcgacagc ggaaccaact cacgttgccg cggcttcctc cggtgcg gtgccctgtc cttttctctc gtccctcctc cccccgtata gttaagcccc ccgctac tactactact agcagcagca gcgctctcgc agcgggagat gcggtgttga 24gcccc gctcggatct cgggactggt gccggctctg cccaggcccc aggctccagg 3ctccct cgacgtttct cggcgagctc gcttgcc atg gag ggc gac gcg gac 355 Met Glu Gly Asp Ala Asp gtg aag tcg ggg agg cgc ggt ggc gga cag gtg tgc cag atc tgc 4Val Lys Ser Gly Arg Arg Gly Gly Gly Gln Val Cys Gln Ile Cys ac ggc gtg ggc acc acg gcg gag ggg gac gtc ttc gcc gcc tgc 45sp Gly Val Gly Thr Thr Ala Glu Gly Asp Val Phe Ala Ala Cys 25 3c gtc tgc ggg ttt ccg gtg tgc cgc ccc tgc tac gag tac gag cgc 499 Asp Val Cys Gly Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg 4 aag gac ggc acg cag gcg tgc ccc cag tgc aag acc aag tac aag cgc 547 Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys Lys Thr Lys Tyr Lys Arg 55 6 cac aag ggg agc ccg gcg atc cgt ggg gag gaa gga gac gac act gat 595 His Lys Gly Ser Pro Ala Ile Arg Gly Glu Glu Gly Asp Asp Thr Asp 75 8c gat agc gac ttc aat tac ctt gca tct ggc aat gag gac cag aag 643 Ala Asp Ser Asp Phe Asn Tyr Leu Ala Ser Gly Asn Glu Asp Gln Lys 9ag att gcc gac aga atg cgc agc tgg cgc atg aac gtt ggg ggc 69ys Ile Ala Asp Arg Met Arg Ser Trp Arg Met Asn Val Gly Gly ggg gat gtt ggt cgc ccc aag tat gac agt ggc gag atc ggg ctt 739 Ser Gly Asp Val Gly Arg Pro Lys Tyr Asp Ser Gly Glu Ile Gly Leu aag tat gac agt ggc gag att cct cgg gga tac atc cca tca gtc 787 Thr Lys Tyr Asp Ser Gly Glu Ile Pro Arg Gly Tyr Ile Pro Ser Val act aac agc cag atc tca gga gaa atc cct ggt gct tcc cct gac cat 835 Thr Asn Ser Gln Ile Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His atg atg tcc cca act ggg aac att ggc aag cgt gct cca ttt ccc 883 His Met Met Ser Pro Thr Gly Asn Ile Gly Lys Arg Ala Pro Phe Pro gtg aac cat tcg cca aat ccg tca agg gag ttc tct ggt agc att 93al Asn His Ser Pro Asn Pro Ser Arg Glu Phe Ser Gly Ser Ile aat gtt gcc tgg aaa gag agg gtt gat ggc tgg aaa atg aag cag 979 Gly Asn Val Ala Trp Lys Glu Arg Val Asp Gly Trp Lys Met Lys Gln 22aag ggg acg att ccc atg acg aat ggc aca agc att gct ccc tct p Lys Gly Thr Ile Pro Met Thr Asn Gly Thr Ser Ile Ala Pro Ser 2225 23gt cgg ggt gtt ggt gat att gat gca tca act gat tac aac atg u Gly Arg Gly Val Gly Asp Ile Asp Ala Ser Thr Asp Tyr Asn Met 235 24aa gat gcc tta ttg aac gac gaa act cga cag cct cta tct agg aaa u Asp Ala Leu Leu Asn Asp Glu Thr Arg Gln Pro Leu Ser Arg Lys 256ca ctt cct tcc tcc agg ata aat cca tac agg atg gtc att gtg l Pro Leu Pro Ser Ser Arg Ile Asn Pro Tyr Arg Met Val Ile Val 265 27tg cga ttg att gtt cta agc atc ttc ttg cac tac cgt atc aca aat u Arg Leu Ile Val Leu Ser Ile Phe Leu His Tyr Arg Ile Thr Asn 289tg cgc aat gca tac cca tta tgg ctt cta tct gtt ata tgt gag o Val Arg Asn Ala Tyr Pro Leu Trp Leu Leu Ser Val Ile Cys Glu 295 33tgg ttt gct ctt tcg tgg ata ttg gat cag ttc cct aag tgg ttt e Trp Phe Ala Leu Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Phe 3325 cca atc aac cgg gag acg tac ctt gat agg ctg gca tta agg tat gac o Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp 334aa ggt gag cca tct cag ttg gct gct gtt gac att ttc gtc agt g Glu Gly Glu Pro Ser Gln Leu Ala Ala Val Asp Ile Phe Val Ser 345 35ca gtc gac cca atg aag gag cct cct ctt gtc act gcc aat acc gtg r Val Asp Pro Met Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val 367cc att ctt gct gtg gat tac cct gtg gat aag gtc tct tgc tat u Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr 375 389ct gat gat gga gct gcg atg ctg aca ttt gat gca cta gct gag l Ser Asp Asp Gly Ala Ala Met Leu Thr Phe Asp Ala Leu Ala Glu 395 4act tca gag ttt gct aga aaa tgg gta cca ttt gtt aag aag tac aac r Ser Glu Phe Ala Arg Lys Trp Val Pro Phe Val Lys Lys Tyr Asn 442aa cct aga gct cct gaa tgg tac ttc tcc cag aaa att gat tac e Glu Pro Arg Ala Pro Glu Trp Tyr Phe Ser Gln Lys Ile Asp Tyr 425 43tg aag gac aaa gtg cac cct tca ttt gtt aaa gac cgc cgg gcc atg u Lys Asp Lys Val His Pro Ser Phe Val Lys Asp Arg Arg Ala Met 445ga gaa tat gaa gaa ttc aaa gtt agg gta aat ggc ctt gtt gct s Arg Glu Tyr Glu Glu Phe Lys Val Arg Val Asn Gly Leu Val Ala 455 467ca cag aaa gtt cct gag gaa gga tgg atc atg caa gat ggc aca s Ala Gln Lys Val Pro Glu Glu Gly Trp Ile Met Gln Asp Gly Thr 475 48ca tgg cca gga aac aat acc agg gac cat cct gga atg att cag gtt o Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val 49ctt ggt cac agt ggt ggc ctt gat act gag ggc aat gag cta ccc e Leu Gly His Ser Gly Gly Leu Asp Thr Glu Gly Asn Glu Leu Pro 55ttg gtc tat gtt tct cgt gaa aag cgt cct gga ttc cag cat cac g Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His 523aa gct ggt gcc atg aat gct ctt gtt cgt gtc tca gct gtg ctt s Lys Ala Gly Ala Met Asn Ala Leu Val Arg Val Ser Ala Val Leu 535 545at gga caa tac atg ttg aat ctt gat tgt gat cac tac att aac 2 Asn Gly Gln Tyr Met Leu Asn Leu Asp Cys Asp His Tyr Ile Asn 555 56ac agt aag gct ctc agg gaa gct atg tgc ttc ctt atg gac cct aac 2 Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Leu Met Asp Pro Asn 578ga agg agt gtc tgc tac gtc cag ttt ccc cag aga ttc gat ggc 2 Gly Arg Ser Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly 585 59tt gac agg aat gat cga tat gcc aac agg aac acc gtg ttt ttc gat 2 Asp Arg Asn Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp 66aac ttg aga ggt ctt gat ggc atc caa gga cca gtt tat gtc gga 2227 Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly 6625 63gc tgt gtt ttc aac cga aca gct cta tat ggt tat gag ccc cca 2275 Thr Gly Cys Val Phe Asn Arg Thr Ala Leu Tyr Gly Tyr Glu Pro Pro 635 64tt aag cag aag aag ggt ggt ttc ttg tca tca cta tgt ggc ggt agg 2323 Ile Lys Gln Lys Lys Gly Gly Phe Leu Ser Ser Leu Cys Gly Gly Arg 656ag gca agc aaa tca aag aag ggc tcg gac aag aag aag tcg cag 237ys Ala Ser Lys Ser Lys Lys Gly Ser Asp Lys Lys Lys Ser Gln 665 67ag cat gtg gac agt tct gtg cca gta ttc aac ctt gaa gat ata gag 24His Val Asp Ser Ser Val Pro Val Phe Asn Leu Glu Asp Ile Glu 689ga gtt gaa ggc gct gga ttt gac gac gag aaa tca ctt ctt atg 2467 Glu Gly Val Glu Gly Ala Gly Phe Asp Asp Glu Lys Ser Leu Leu Met 695 77caa atg agc ctg gag aag aga ttt ggc cag tcc gca gcg ttt gtt 25Gln Met Ser Leu Glu Lys Arg Phe Gly Gln Ser Ala Ala Phe Val 7725 gcc tcc act ctg atg gag tat ggt ggt gtt cct cag tcc gca act ccg 2563 Ala Ser Thr Leu Met Glu Tyr Gly Gly Val Pro Gln Ser Ala Thr Pro 734ct ctt ctg aaa gaa gct atc cat gtt ata agc tgt ggc tat gag 26Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu 745 75ac aag act gaa tgg gga act gag atc ggg tgg atc tac ggt tct gtg 2659 Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly Trp Ile Tyr Gly Ser Val 767aa gac att ctc acc gga ttc aag atg cac gcg cga ggc tgg cgg 27Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly Trp Arg 775 789tc tac tgc atg ccc aag cgg cca gct ttc aag ggg tct gcc ccc 2755 Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro 795 8atc aat ctt tcg gac cgt ctg aac cag gtg ctc cgg tgg gct ctt ggg 28Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala Leu Gly 882tg gag atc ctc ttc agc cgg cac tgc ccc ctg tgg tac ggc tac 285al Glu Ile Leu Phe Ser Arg His Cys Pro Leu Trp Tyr Gly Tyr 825 83ga ggg cgg ctc aag ttc ctg gag aga ttc gcg tac atc aac acc acc 2899 Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe Ala Tyr Ile Asn Thr Thr 845ac ccg ctc acg tcc atc ccg ctt ctc atc tac tgc atc ctg ccc 2947 Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu Ile Tyr Cys Ile Leu Pro 855 867tc tgt ctg ctc acc gga aag ttc atc att cca gag atc agc aac 2995 Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Glu Ile Ser Asn 875 88tc gcc agc atc tgg ttc atc tcc ctc ttc atc tcg atc ttc gcc acg 3 Ala Ser Ile Trp Phe Ile Ser Leu Phe Ile Ser Ile Phe Ala Thr 89atc ctg gag atg agg tgg agc ggg gtg ggc atc gac gag tgg tgg 3 Ile Leu Glu Met Arg Trp Ser Gly Val Gly Ile Asp Glu Trp Trp 99aac gag cag ttc tgg gtg atc ggg ggc atc tcc gcg cac ctc ttc 3 Asn Glu Gln Phe Trp Val Ile Gly Gly Ile Ser Ala His Leu Phe 923tg ttc cag ggc ctg ctc aag gtg ctg gcc ggc atc gac acc aac 3 Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp Thr Asn 935 945cc gtc acc tcc aag gcc tcg gac gag gac ggc gac ttc gcg gag 3235 Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe Ala Glu 955 96tg tac atg ttc aag tgg acg acg ctc ctg atc ccg ccc acc acc atc 3283 Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Ile 978tc atc aac ctg gtc ggc gtc gtc gcc ggc atc tcc tac gcc atc 333le Ile Asn Leu Val Gly Val Val Ala Gly Ile Ser Tyr Ala Ile 985 99ac agc gga tac cag tcg tgg ggc ccg ctc ttc ggc aag ctc ttc ttc 3379 Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe gcc ttc tgg gtc atc gtc cac ctg tac ccg ttc ctc aag ggc ctc atg 3427 Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met 2c agg cag aac cgc acc ccg acc atc gtc gtc gtc tgg gcc atc ctg 3475 Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Val Val Trp Ala Ile Leu 4ctg gcg tcc atc ttc tcc ttg ctg tgg gtt cgc atc gac ccc ttc acc 3523 Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Thr 55 c cgc gtc act ggc ccg gat acc cag acg tgt ggc atc aac tgc 3568 Thr Arg Val Thr Gly Pro Asp Thr Gln Thr Cys Gly Ile Asn Cys 7tagggaagtg gaaggtttgt actttgtaga aacggaggaa taccacgtgc catctgttgt 3628 ctgttaagtt atatatatat aagcagcaag tggcgttatt tacagctacg tacagaccag 3688 tggatattgt ttaccacaaa gttttacttg tgttaatatg cattcttttg ttgatataaa 3748 aaaaaaaaaa aaaaagggcg gccgc 3773 6 T Zea mays 6 Met Glu Gly Asp Ala Asp Gly Val Lys Ser Gly Arg Arg Gly Gly Gly Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly 2 Asp Val Phe Ala Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro 35 4s Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys 5 Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Ala Ile Arg Gly Glu 65 7 Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe Asn Tyr Leu Ala Ser 85 9y Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp Arg Met Arg Ser Trp Met Asn Val Gly Gly Ser Gly Asp Val Gly Arg Pro Lys Tyr Asp Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser Gly Glu Ile Pro Arg Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His His Met Met Ser Pro Thr Gly Asn Ile Gly >
Lys Arg Ala Pro Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg Phe Ser Gly Ser Ile Gly Asn Val Ala Trp Lys Glu Arg Val Asp 2Trp Lys Met Lys Gln Asp Lys Gly Thr Ile Pro Met Thr Asn Gly 222er Ile Ala Pro Ser Glu Gly Arg Gly Val Gly Asp Ile Asp Ala 225 234hr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg 245 25ln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser Ser Arg Ile Asn Pro 267rg Met Val Ile Val Leu Arg Leu Ile Val Leu Ser Ile Phe Leu 275 28is Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu Trp Leu 29Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp 33Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg 325 33eu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Ala 345sp Ile Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro Leu 355 36al Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val 378ys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr 385 39Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp Val Pro 44Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe 423ln Lys Ile Asp Tyr Leu Lys Asp Lys Val His Pro Ser Phe Val 435 44ys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg 456sn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp 465 478et Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His 485 49ro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr 55Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 5525 Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val 534al Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp 545 556sp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys 565 57he Leu Met Asp Pro Asn Leu Gly Arg Ser Val Cys Tyr Val Gln Phe 589ln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg 595 6Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln 662ro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu 625 634ly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe Leu Ser 645 65er Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys Ser Lys Lys Gly Ser 667ys Lys Lys Ser Gln Lys His Val Asp Ser Ser Val Pro Val Phe 675 68sn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe Asp Asp 69Lys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg Phe Gly 77Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val 725 73ro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val 745er Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly 755 76rp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 778la Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala 785 79Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val 88Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Phe Ser Arg His Cys 823eu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe 835 84la Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu 856yr Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile 865 878ro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser Leu Phe 885 89le Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val 99Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly 9925 Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu 934ly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu 945 956ly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu 965 97le Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala 989le Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu 995 Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val 3l Val Trp Ala Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val 5Arg Ile Asp Pro Phe Thr Thr Arg Val Thr Gly Pro Asp Thr Gln Thr 65 s Gly Ile Asn Cys 25 DNA Zea mays 7 atggagggcg acgcggacgg cgtga 25 8 25 DNA Zea mays 8 ctagcagttg atgccacacg tctgg 25 9 378ea mays CDS (23425) 9 gtcgacccac gcgtccgcag cagcagaagc actgcgcggc attgcagcga tcgagcggga 6ttggg gcatggtggt cgccaacgcc gctcggatct agaggcccgc acgggccgat tctccgc ccgcctcgtc ggtgttggtg tcgttggcgt gtggagccgt ctcggtggga gcgggga gggagcggag atg gcg gcc aac aag ggg atg gtg gcg ggc tcg 233 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser cac aac cgc aac gag ttc gtc atg atc cgc cac gac ggc gat gtg ccg 28sn Arg Asn Glu Phe Val Met Ile Arg His Asp Gly Asp Val Pro 5 ggc tcg gct aag ccc aca aag agt gcg aat gga cag gtc tgc cag att 329 Gly Ser Ala Lys Pro Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile 3 tgc ggt gac tct gtg ggt gtt tca gcc act ggt gat gtc ttt gtt gcc 377 Cys Gly Asp Ser Val Gly Val Ser Ala Thr Gly Asp Val Phe Val Ala 45 5c aat gag tgt gcc ttc cct gtc tgc cgc cca tgc tat gag tat gag 425 Cys Asn Glu Cys Ala Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu 6 75 cgc aag gag ggg aac caa tgc tgc ccc cag tgc aag act aga tac aag 473 Arg Lys Glu Gly Asn Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys 8 aga cag aaa ggt agc cct cga gtt cat ggt gat gag gat gag gaa gat 52ln Lys Gly Ser Pro Arg Val His Gly Asp Glu Asp Glu Glu Asp 95 gtt gat gac cta gac aat gaa ttc aac tac aag caa ggc agt ggg aaa 569 Val Asp Asp Leu Asp Asn Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys cca gag tgg caa ctg caa gga gat gat gct gat ctg tct tca tct 6Pro Glu Trp Gln Leu Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser cgc cat gag cca cat cat cgg att cca cgc ctg aca agc ggt caa 665 Ala Arg His Glu Pro His His Arg Ile Pro Arg Leu Thr Ser Gly Gln cag ata tct gga gag att cct gat gct tcc cct gac cgt cat tct atc 7Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile agt cca aca tcg agc tat gtt gat cca agc gtc cca gtt cct gtg 76er Pro Thr Ser Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val att gtg gac ccc tcg aag gac ttg aat tcc tat ggg ctt aat agt 8Ile Val Asp Pro Ser Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser 2gac tgg aag gaa aga gtt gag agc tgg agg gtt aaa cag gac aaa 857 Val Asp Trp Lys Glu Arg Val Glu Ser Trp Arg Val Lys Gln Asp Lys 22atg atg caa gtg act aat aaa tat cca gag gct aga gga gga gac 9Met Met Gln Val Thr Asn Lys Tyr Pro Glu Ala Arg Gly Gly Asp 223tg gag ggg act ggc tca aat gga gaa gat atg caa atg gtt gat gat 953 Met Glu Gly Thr Gly Ser Asn Gly Glu Asp Met Gln Met Val Asp Asp 245gg cta cct ttg agc cgt atc gtg cca att tcc tca aac cag ctc a Arg Leu Pro Leu Ser Arg Ile Val Pro Ile Ser Ser Asn Gln Leu 255 26ac ctt tac cgg gta gtg atc att ctc cgt ctt atc atc ctg tgc ttc n Leu Tyr Arg Val Val Ile Ile Leu Arg Leu Ile Ile Leu Cys Phe 278tc cag tat cgt gtc agt cat cca gtg cgt gat gct tat gga tta e Phe Gln Tyr Arg Val Ser His Pro Val Arg Asp Ala Tyr Gly Leu 285 29gg cta gta tct gtt atc tgc gag gtc tgg ttt gcc ttg tct tgg ctt p Leu Val Ser Val Ile Cys Glu Val Trp Phe Ala Leu Ser Trp Leu 33cta gat cag ttc cca aaa tgg tat cca atc aac cgt gag aca tat ctt u Asp Gln Phe Pro Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu 323gg ctt gca ttg agg tat gat aga gag gga gag cca tca cag ctg p Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu 335 34ct ccc att gat gtc ttc gtc agt aca gtg gat cca ttg aag gaa cct a Pro Ile Asp Val Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro 356tg atc aca gcc aac act gtt ttg tcc att ctt tct gtg gat tac o Leu Ile Thr Ala Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr 365 37ct gtt gac aaa gtg tca tgc tat gtt tct gat gat ggt tca gct atg o Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ser Ala Met 389tg act ttt gag tct ctc tca gaa acc gca gaa ttt gct aga aag tgg u Thr Phe Glu Ser Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp 44ccc ttt tgt aag aag cac aat att gaa cca aga gct cca gaa ttt l Pro Phe Cys Lys Lys His Asn Ile Glu Pro Arg Ala Pro Glu Phe 4425 tac ttt gct caa aaa ata gat tac ctg aag gac aaa att caa cct tca r Phe Ala Gln Lys Ile Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser 434tt aag gaa aga cgc gca atg aag agg gag tat gaa gaa ttc aaa e Val Lys Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys 445 45ta aga atc aat gcc ctt gtt gcc aaa gca cag aaa gtg cct gaa gag l Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu 467gg tgg acc atg gct gat gga act gca tgg cct ggg aat aat cct agg y Trp Thr Met Ala Asp Gly Thr Ala Trp Pro Gly Asn Asn Pro Arg 489at cct ggc atg att cag gtt ttc ttg ggg cac agt ggt ggg ctc p His Pro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu 495 5gac act gat gga aat gag tta cca cgt ctt gtc tat gtc tct cgt gaa p Thr Asp Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu 552ga cca ggc ttt cag cat cac aag aag gct ggt gca atg aat gcg s Arg Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala 525 53tg att cgt gta tct gct gtg ctg aca aat ggt gcc tat ctt ctc aat u Ile Arg Val Ser Ala Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn 545tg gat tgc gac cat tac ttc aat agc agc aaa gct ctt aga gaa gca l Asp Cys Asp His Tyr Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala 567gc ttc atg atg gat ccg gct cta gga agg aaa act tgt tat gta t Cys Phe Met Met Asp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val 575 58aa ttt cca cag aga ttt gat ggc att gac ttg cac gat cga tat gct 2 Phe Pro Gln Arg Phe Asp Gly Ile Asp Leu His Asp Arg Tyr Ala 59cgg aac ata gtt ttc ttt gat atc aac atg aaa ggt ctg gat ggc 2 Arg Asn Ile Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly 66cag ggt cca gtt tac gtg gga aca gga tgc tgt ttc aat aga cag 2 Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln 623ct ttg tat gga tac gat cct gtt ttg act gaa gct gat ctg gag cca 2 Leu Tyr Gly Tyr Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro 645tt gtt att aag agc tgc tgt ggt aga agg aag aaa aag aac aag 22Ile Val Ile Lys Ser Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys 655 66gt tat atg gat agt caa agc cgt att atg aag aga aca gaa tct tca 2249 Ser Tyr Met Asp Ser Gln Ser Arg Ile Met Lys Arg Thr Glu Ser Ser 678cc atc ttc aat atg gaa gac atc gaa gag ggt att gaa ggt tac 2297 Ala Pro Ile Phe Asn Met Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr 685 69ag gat gaa agg tca gtg ctt atg tcc cag agg aaa ttg gag aaa cgc 2345 Glu Asp Glu Arg Ser Val Leu Met Ser Gln Arg Lys Leu Glu Lys Arg 77ttt ggt cag tct cct att ttc att gca tcc acc ttt atg aca caa ggt 2393 Phe Gly Gln Ser Pro Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly 723ta cca cct tca aca aac cca gct tct cta cta aag gaa gct atc 244le Pro Pro Ser Thr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile 735 74at gtc atc agt tgt gga tat gag gac aaa act gaa tgg gga aaa gag 2489 His Val Ile Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu 756gc tgg atc tat ggt tca gta acg gag gat att ctg act ggg ttt 2537 Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe 765 77aa atg cat gca agg ggc tgg caa tca atc tac tgc atg cca cca cga 2585 Lys Met His Ala Arg Gly Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg 789ct tgt ttc aag ggt tct gca cca atc aat ctt tcc gat cgt ctt aat 2633 Pro Cys Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn 88gtg ctc cgt tgg gct ctt ggg tca gtg gaa att ctg ctt agt aga 268al Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Leu Ser Arg 8825 cat tgt cct atc tgg tat ggt tac aat gga cga ttg aag ctt ttg gag 2729 His Cys Pro Ile Trp Tyr Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu 834tg gct tac atc aac act att gta tat cca atc aca tcc att ccg 2777 Arg Leu Ala Tyr Ile Asn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro 845 85tt att gcc tat tgt gtg ctt ccc gct atc tgc ctc ctt acc aat aaa 2825 Leu Ile Ala Tyr Cys Val Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys 867tt atc att cct gag att agc aat tat gct ggg atg ttc ttc att ctt 2873 Phe Ile Ile Pro Glu Ile Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu 889tc gcc tcc att ttt gcc act ggt ata ttg gag ctt aga tgg agt 292he Ala Ser Ile Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser 895 9ggt gtt ggc att gaa gat tgg tgg aga aat gag cag ttt tgg gtt att 2969 Gly Val Gly Ile Glu Asp Trp Trp Arg Asn Glu
Gln Phe Trp Val Ile 992gc acc tct gcc cat ctc ttc gca gtg ttc cag ggt ctg ctg aaa 3 Gly Thr Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys 925 93tg ttg gct ggg att gat acc aac ttc aca gtt acc tca aag gca tct 3 Leu Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser 945at gag gat ggc gac ttt gct gag cta tat gtg ttc aag tgg acc agt 3 Glu Asp Gly Asp Phe Ala Glu Leu Tyr Val Phe Lys Trp Thr Ser 967tc att cct ccg acc act gtt ctt gtc att aac ctg gtc gga atg 3 Leu Ile Pro Pro Thr Thr Val Leu Val Ile Asn Leu Val Gly Met 975 98tg gca gga att tct tat gcc att aac agt ggc tac caa tcc tgg ggt 32Ala Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly 99 ctc ttt gga aag ctg ttc ttc tcg atc tgg gtg atc ctc cat ctc 3257 Pro Leu Phe Gly Lys Leu Phe Phe Ser Ile Trp Val Ile Leu His Leu tac ccc ttc ctc aag ggt ctc atg gga agg cag aac cgc aca cca aca 33Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr 25 35 atc gtc att gtc tgg tcc atc ctt ctt gca tct atc ttc tcc ttg ctg 3353 Ile Val Ile Val Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu 45 g gtg aag atc gat cct ttc atc tcc ccg aca cag aaa gct gct gcc 34Val Lys Ile Asp Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala 6ttg ggg caa tgt ggc gtc aac tgc tgatcgagac agtgactctt atttgaagag 3455 Leu Gly Gln Cys Gly Val Asn Cys 75 gctcaatcaa gatctgcccc ctcgtgtaaa tacctgagga ggctagatgg gaattccttt 35gtaggt gaggatggat ttgcatctaa gttatgcctc tgttcattag cttcttccgt 3575 gccggtgctg ctgcggacta agaatcacgg agcctttcta ccttccatgt agcgccagcc 3635 agcagcgtaa gatgtgaatt ttgaagtttt gttatgcgtg cagtttattg ttttagagta 3695 aattatcatt tgtttgtggg aactgttcac acgagcttat aatggcaatg ctgttattta 3755 aaaaaaaaaa aaaaagggcg gccgc 37875 PRT Zea mays Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu Val Met Ile Arg His Asp Gly Asp Val Pro Gly Ser Ala Lys Pro 2 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Ser Val 35 4y Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9o Arg Val His Gly Asp Glu Asp Glu Glu Asp Val Asp Asp Leu Asp Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys Gly Pro Glu Trp Gln Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Glu Pro His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Met Gln Val 222sn Lys Tyr Pro Glu Ala Arg Gly Gly Asp Met Glu Gly Thr Gly 225 234sn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu 245 25er Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Val 267le Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg 275 28al Ser His Pro Val Arg Asp Ala Tyr Gly Leu Trp Leu Val Ser Val 29Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro 33Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu 325 33rg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val 345al Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala 355 36sn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp Lys Val 378ys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser 385 39Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys 44His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys 423sp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg 435 44rg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala 456al Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala 465 478ly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met 485 49le Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn 55Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe 5525 Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser 534al Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His 545 556he Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met 565 57sp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg 589sp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val 595 6Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val 662al Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr 625 634ro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Ile Lys 645 65er Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys Ser Tyr Met Asp Ser 667er Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn 675 68et Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser 69Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro 77Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser 725 73hr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys 745yr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr 755 76ly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg 778rp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly 785 79Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp 88Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp 823ly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile 835 84sn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Ile Ala Tyr Cys 856eu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu 865 878er Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile 885 89he Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu 99Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala 9925 His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile 934hr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp 945 956la Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro 965 97hr Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser 989la Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys 995 Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp 3r Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp 5Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly 65 l Asn Cys 25 DNA Zea mays cggcca acaaggggat ggtgg 25 NA Zea mays cagttg acgccacatt gcccc 25 DNA Zea mays CDS ((34gcagcagcag caccaccact gcgcggcatt gcagcgagca agcgggaggg atctggggca 6gcggt cgctgccgct gccgctcgga tctagagggc cgcacgggct gattgccctc cggcctc gtcggtgtcg gtggagtgtg aatcggtgtg tgtaggagga gcgcggag gcg gcc aac aag ggg atg gtg gca ggc tct cac aac cgc aac gag 226 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu gtc atg atc cgc cac gac ggc gac gcg cct gtc ccg gct aag ccc 274 Phe Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 2 acg aag agt gcg aat ggg cag gtc tgc cag att tgt ggc gac act gtt 322 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 4c gtt tca gcc act ggt gat gtc ttt gtt gcc tgc aat gag tgt gcc 37al Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 ttc cct gtc tgc cgc cct tgc tat gag tac gag cgc aag gaa ggg aac 4Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 caa tgc tgc cct cag tgc aag act aga tac aag aga cag aaa ggt agc 466 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9t cga gtt cat ggt gat gat gag gag gaa gat gtt gat gac ctg gac 5Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp gaa ttc aac tat aag caa ggc aat ggg aag ggc cca gag tgg cag 562 Asn Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln caa gga gat gac gct gat ctg tct tca tct gct cgc cat gac cca 6Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro cat cgg att cca cgc ctt aca agt gga caa cag ata tct gga gag 658 His His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu atc cct gat gca tcc cct gac cgt cat tct atc cgc agt cca aca tcg 7Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser tat gtt gat cca agc gtt cca gtt cct gtg agg att gtg gac ccc 754 Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro aag gac ttg aat tcc tat ggg ctt aat agt gtt gac tgg aag gaa 8Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2gtt gag agc tgg agg gtt aaa cag gac aaa aat atg ttg caa gtg 85al Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 222at aaa tat cca gag gct aga gga gac atg gag ggg act ggc tca 898 Thr Asn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser 225 234ga gaa gat atg caa atg gtt gat gat gca cgc cta cct ttg agc 946 Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 25gc att gtg cca att tcc tca aac cag ctc aac ctt tac cgg ata gta 994 Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 267tt ctc cgt ctt atc atc ctg tgc ttc ttc ttc caa tat cgt atc e Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 28gt cat cca gtg cgt aat gct tat gga ttg tgg cta gta tct gtt atc r His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 29gag gtc tgg ttt gcc ttg tcc tgg ctt cta gat cag ttc cca aaa s Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys 33tgg tat cca atc aac cgt gag aca tat ctc gac agg ctt gca ttg agg p Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 33at gat aga gag gga gag cca tca cag ctg gct ccc att gat gtc ttt r Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 345gt aca gtg gat cca ttg aag gaa cct cca ctg atc aca gcc aac l Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 36ct gtt ttg tcc att ctt gct gtg gat tac cct gtt gac aaa gtg tca r Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 378at gtt tct gat gat ggc tca gct atg ctg act ttt gag tct ctc s Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu 385 39gaa act gcc gaa ttt gct aga aag tgg gtt ccc ttt tgt aag aag r Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 44aat att gaa cca aga gct cca gaa ttt tac ttt gct caa aaa ata s Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 423ac ctg aag gac aaa att caa cct tca ttt gtt aag gaa aga cga p Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 44ca atg aag aga gag tat gaa gaa ttc aaa ata aga atc aat gcc ctt a Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 456cc aaa gca cag aaa gtg cct gaa gag ggg tgg acc atg gct gat l Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp 465 478ct gct tgg cct ggg aat aac cct agg gac cat cct ggc atg att y Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 49ag gtg ttc ttg ggg cac agt ggt ggg ctt gac act gat gga aat gaa n Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 55cca cgt ctt gtc tat gtc tct cgt gaa aag aga cca ggc ttt cag u Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln 5525 cat cac aag aag gct ggt gca atg aat gca ctg att cgt gta tct gct s His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 534tg aca aat ggt gcc tat ctt ctc aat gtg gat tgt gac cat tac l Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr 545 556at agc agc aaa gct ctt aga gaa gca atg tgc ttc atg atg gat e Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 57ca gct cta gga agg aaa act tgt tat gta caa ttt cca caa aga ttt o Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 589gc att gac ttg cac gat cga tat gct aat agg aac ata gtc ttc 2 Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 6ttt gat atc aac atg aaa ggt cta gat ggc att cag ggt cca gtc tat 2>
Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 662ga aca gga tgc tgt ttc aat agg cag gct ttg tat gga tat gat 2 Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp 625 634tt ttg act gaa gct gat ctg gaa cct aac att gtt gtt aag agc 2 Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 65gc tgt ggt aga agg aag aga aag aac aag agt tat atg gat agt caa 2 Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 667gt att atg aag aga aca gaa tct tca gct ccc atc ttt aac atg 2242 Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 68aa gac atc gag gag ggt att gaa ggt tat gag gat gaa agg tca gtg 229sp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 69atg tcc cag agg aaa ttg gag aaa cgc ttt ggt cag tct cca atc 2338 Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile 77ttc att gca tcc acc ttt atg act caa ggt ggc ata cca cct tca aca 2386 Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 73ac cca gct tct cta ctg aag gaa gct atc cat gtt atc agc tgt ggg 2434 Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 745ag gac aaa act gaa tgg gga aaa gag att ggc tgg atc tat ggt 2482 Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 76ca gtt aca gag gat att ctg act ggg ttt aaa atg cat gca aga ggc 253al Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 778aa tca atc tac tgc atg cca cca cga cct tgt ttc aag ggt tct 2578 Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser 785 79cca atc aat ctt tct gat cgt ctt aat cag gtg ctc cgt tgg gct 2626 Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 88ggg tca gtg gaa att ctg ctt agc aga cat tgt cct ata tgg tat 2674 Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 823ac aat ggg cga ttg aag ctt ttg gag agg ctg gct tac att aac 2722 Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 84cc att gtt tat cca atc aca tct gtt ccg ctt atc gcc tat tgt gtg 277le Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 856ct gct atc tgt ctt ctt acc aat aaa ttt atc att cct gag att 28Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile 865 878at tat gct gga atg ttc ttc att ctt ctt ttt gcc tcc att ttc 2866 Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 89ca act ggt ata ttg gag ctc aga tgg agt ggt gtt ggc att gaa gat 29Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 99tgg aga aat gag cag ttt tgg gtt att ggt ggc acc tct gcc cat 2962 Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 9925 ctc ttc gcg gtg ttc cag ggt ctg ctg aaa gtg ttg gct ggg att gat 3 Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 934ac ttc aca gtt acc tca aag gca tct gat gag gat ggc gac ttt 3 Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 945 956ag cta tat gtg ttc aag tgg acc agt ttg ctc atc cct ccg acc 3 Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 97ct gtt ctt gtc att aac ctg gtc gga atg gtg gca gga att tcg tat 3 Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 989tt aac agc ggc tac caa tcc tgg ggt ccg ctc ttt gga aag ctg 32Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 ttc tcg atc tgg gtg atc ctc cat ctc tac ccc ttc ctc aag ggt 325he Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly ctc atg ggc agg cag aac cgc acg cca aca atc gtc atc gtt tgg tcc 3298 Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser 3c ctc ctt gcg tct atc ttc tcc ttg ctg tgg gtg aag atc gat cct 3346 Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 5ttc atc tcc ccg aca cag aaa gct gcc gcc ttg ggg caa tgt ggt gtg 3394 Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 65 c tgc tgatccagat tgtgactctt atctgaagag gctcagccaa agatctgccc 345ys cctcgtgtaa atacctgagg gggctagatg ggaatttttt gttgtagatg aggatggatc 35tccaag ttatgcctct gtttattagc ttcttcggtg ccggtgctgc tgcagacaat 357agcct ttctaccttg cttgtagtgc tggccagcag cgtaaattgt gaattctgca 363ttata cgtggtgttt attgttttag agtaaattat catttgtttg aggtaactat 369cgaac tatatggcaa tgctgttatt taaaa 3725 PRT Zea mays Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 2 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 4y Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9o Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 222sn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser 225 234ly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 25rg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 267le Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 28er His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 29Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys 33Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 33yr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 345er Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 36hr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 378yr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu 385 39Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 44Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 423yr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 44la Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 456la Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp 465 478hr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 49ln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 55Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln 5525 His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 534eu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr 545 556sn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 57ro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 589ly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 6Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 662ly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp 625 634al Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 65ys Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 667rg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 68lu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 69Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile 77Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 73sn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 745lu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 76er Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 778ln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser 785 79Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 88Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 823yr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 84hr Ile Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 856ro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile 865 878sn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 89la Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 99Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 9925 Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 934sn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 945 956lu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 97hr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 989le Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser 3e Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 5Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 65 n Cys NA Zea mays cggcca acaaggggat ggtgg 25 NA Zea mays cagttc acaccacatt gcccc 25 DNA Zea mays CDS ((34cttctccctc gtcggtgcgg cgtggcgcgg ctcggcgttc ggtgagaaac cactcggggg 6gatct gctgctagag tgagaggagc tacggtcagt atcctctgcc ttcgtcggcg gaagtgg aggggaggaa gcg atg gag gcg agc gcc ggg ctg gtg gcc ggc Glu Ala Ser Ala Gly Leu Val Ala Gly tcc cac aac cgc aac gag ctc gtc gtc atc cgc cgc gac ggc gat ccc 22is Asn Arg Asn Glu Leu Val Val Ile Arg Arg Asp Gly Asp Pro 5 ggg ccg aag ccg ccg cgg gag cag aac ggg cag gtg tgc cag att tgc 269 Gly Pro Lys Pro Pro Arg Glu Gln Asn Gly Gln Val Cys Gln Ile Cys 3 ggc gac gac gtc ggc ctt gcc ccc ggc ggg gac ccc ttc gtg gcg tgc 3Asp Asp Val Gly Leu Ala Pro Gly Gly Asp Pro Phe Val Ala Cys 45 5c gag tgc gcc ttc ccc gtc tgc cgg gac tgc tac gaa tac gag cgc 365 Asn Glu Cys Ala Phe Pro Val Cys Arg Asp Cys Tyr Glu Tyr Glu Arg 6 cgg gag ggc acg cag aac tgc ccc cag tgc aag act cga tac aag cgc 4Glu Gly Thr Gln Asn Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg 75 8 ctc aag ggc tgc caa cgt gtg acc ggt gac gag gag gag gac ggc gtc 46ys Gly Cys Gln Arg Val Thr Gly Asp Glu Glu Glu Asp Gly Val 95 gat gac ctg gac aac gag ttc aac tgg gac ggc cat gac tcg cag tct 5Asp Leu Asp Asn Glu Phe Asn Trp Asp Gly His Asp Ser Gln Ser gcc gag tcc atg ctc tac ggc cac atg agc tac ggc cgt gga ggt 557 Val Ala Glu Ser Met Leu Tyr Gly His Met Ser Tyr Gly Arg Gly Gly cct aat ggc gcg cca caa gct ttc cag ctc aac ccc aat gtt cca 6Pro Asn Gly Ala Pro Gln Ala Phe Gln Leu Asn Pro Asn Val Pro ctc acc aac ggg caa atg gtg gat gac atc cca ccg gag cag cac 653 Leu Leu Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His gcg ctg gtg cct tct ttc atg ggt ggt ggg gga aag agg ata cat ccc 7Leu Val Pro Ser Phe Met Gly Gly Gly Gly Lys Arg Ile His Pro cct tat gcg gat ccc agc tta cct gtg caa ccc agg tct atg gac 749 Leu Pro Tyr Ala Asp Pro Ser Leu Pro Val Gln Pro Arg Ser Met Asp 2tcc aag gat ctt gct gca tat ggg tat ggt agt gtt gct tgg aag 797 Pro Ser Lys Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp Lys 22cgg atg gag aat tgg aag cag aga caa gag agg atg cac cag acg 845 Glu Arg Met Glu Asn Trp Lys Gln Arg Gln Glu Arg Met His Gln Thr 223at gat ggt ggt ggt gat gat ggt gac gat gct gat cta cca cta 893 Gly Asn Asp Gly Gly Gly Asp Asp Gly Asp Asp Ala Asp Leu Pro Leu 235 245at gaa gca aga caa caa ctg tcc agg aaa att cca ctt cca tca 94sp Glu Ala Arg Gln Gln Leu Ser Arg Lys Ile Pro Leu Pro Ser 255 26gc cag att aat cca tat agg atg att atc att att cgg ctt gtg gtt 989 Ser Gln Ile Asn Pro Tyr Arg Met Ile Ile Ile Ile Arg Leu Val Val 278gg ttc ttc ttc cac tac cga gtg atg cat ccg gtg aat gat gca u Gly Phe Phe Phe His Tyr Arg Val Met His Pro Val Asn Asp Ala 285 29tt gct ttg tgg ctc ata tct gtt atc tgt gaa atc tgg ttt gcc atg e Ala Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe Ala Met 33tgg att ctt gat caa ttc cca aag tgg ttc cct
att gag aga gag r Trp Ile Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu 3325 33ac cta gac cgg ctg tca ctg agg ttc gac aag gaa ggc cag cca r Tyr Leu Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly Gln Pro 335 34ct caa ctt gct cca att gat ttc ttt gtc agt acg gtt gat ccc tta r Gln Leu Ala Pro Ile Asp Phe Phe Val Ser Thr Val Asp Pro Leu 356aa cct cct ttg gtc aca aca aat act gtt cta tct atc ctt tcg s Glu Pro Pro Leu Val Thr Thr Asn Thr Val Leu Ser Ile Leu Ser 365 37tg gat tat cct gtt gat aag gtt tct tgc tat gtt tct gat gat ggt l Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly 389ca atg cta acg ttt gaa gca tta tct gaa aca tct gaa ttt gca a Ala Met Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala 395 44aaa tgg gtt cct ttc tgc aaa cgg tac aat att gaa cct cgc gct s Lys Trp Val Pro Phe Cys Lys Arg Tyr Asn Ile Glu Pro Arg Ala 4425 cca gag tgg tac ttc caa cag aag ata gac tac ttg aaa gac aag gtg o Glu Trp Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val 434ca aac ttt gtt agg gag agg aga gca atg aag aga gag tat gag a Ala Asn Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu 445 45aa ttc aag gtg aga atc aat gcc tta gtt gcc aaa gcc cag aaa gtt u Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Val 467aa gaa gga tgg aca atg caa gat gga acc ccc tgg cct gga aac o Glu Glu Gly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn 475 489tt cgt gat cat cct gga atg att cag gtc ttc ctt ggc caa agc n Val Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly Gln Ser 495 5gga ggc ctt gac tgt gag gga aat gaa ctg cca cga ttg gtt tat gtt y Gly Leu Asp Cys Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val 552ga gag aaa cga cca ggc tat aac cat cat aag aaa gct ggt gct r Arg Glu Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala Gly Ala 525 53tg aat gca ttg gtc cga gtc tct gct gta cta aca aat gct cca tat t Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr 545ta aac ttg gat tgt gat cac tac atc aac aac agc aag gct ata u Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Ile 555 567aa gca atg tgt ttt atg atg gac cct tta cta gga aag aag gtt s Glu Ala Met Cys Phe Met Met Asp Pro Leu Leu Gly Lys Lys Val 575 58gc tat gta cag ttc cct caa aga ttt gat ggg att gat cgc cat gac s Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg His Asp 59tat gct aac cgg aat gtt gtc ttt ttt gat atc aac atg aaa ggt g Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met Lys Gly 66gat ggt att cag ggt cca att tat gtt ggt act gga tgt gta ttt 2 Asp Gly Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys Val Phe 623gg cag gca tta tat ggt tat gat gcc ccc aaa aca aag aag cca 2 Arg Gln Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro 635 645ca agg act tgc aac tgc tgg ccc aag tgg tgc ttt tgc tgt tgc 2 Ser Arg Thr Cys Asn Cys Trp Pro Lys Trp Cys Phe Cys Cys Cys 655 66gc ttt ggc aat agg aag caa aag aag act acc aaa ccc aaa aca gag 2 Phe Gly Asn Arg Lys Gln Lys Lys Thr Thr Lys Pro Lys Thr Glu 678aa aag tta tta ttt ttc aag aaa gaa gag aac caa tcc cct gca 2237 Lys Lys Lys Leu Leu Phe Phe Lys Lys Glu Glu Asn Gln Ser Pro Ala 685 69at gct ctt ggt gaa att gac gaa gct gct cca gga gct gag aat gaa 2285 Tyr Ala Leu Gly Glu Ile Asp Glu Ala Ala Pro Gly Ala Glu Asn Glu 77gcc ggt att gta aat caa caa aaa tta gaa aag aaa ttt ggc caa 2333 Lys Ala Gly Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln 7725 73ct gtt ttt gtt aca tcc aca ctt ctc gag aat ggt gga acc ttg 238er Val Phe Val Thr Ser Thr Leu Leu Glu Asn Gly Gly Thr Leu 735 74ag agt gca agt cct gct tct ctt ttg aaa gaa gct ata cat gtc att 2429 Lys Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile 756gt ggt tat gaa gac aag aca gac tgg gga aaa gag att ggc tgg 2477 Ser Cys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Lys Glu Ile Gly Trp 765 77tc tat gga tca gtt aca gaa gat att cta act ggt ttc aag atg cat 2525 Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His 789at ggt tgg cgg tca att tac tgc ata cct aaa cgg gtt gca ttc 2573 Cys His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Val Ala Phe 795 88ggt tct gca cct ctg aat ctt tca gat cgt ctt cac cag gtg ctt 262ly Ser Ala Pro Leu Asn Leu Ser Asp Arg Leu His Gln Val Leu 8825 cgg tgg gct ctt ggg tct att gag atc ttc ttc agc aat cat tgc cct 2669 Arg Trp Ala Leu Gly Ser Ile Glu Ile Phe Phe Ser Asn His Cys Pro 834gg tat ggg tat ggt ggc ggt ctg aaa ttt ttg gaa aga ttt tcc 27Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser 845 85ac atc aac tcc atc gtg tat cct tgg aca tct att ccc ctc ttg gct 2765 Tyr Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala 867gt aca ttg cct gcc atc tgt tta ttg aca ggg aaa ttt atc act 28Cys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr 875 889ag ctg aat aat gtt gcc agc ctg tgg ttc atg tca ctt ttt atc 286lu Leu Asn Asn Val Ala Ser Leu Trp Phe Met Ser Leu Phe Ile 895 9tgc att ttt gct acg agc atc cta gaa atg aga tgg agt ggt gtt gga 29Ile Phe Ala Thr Ser Ile Leu Glu Met Arg Trp Ser Gly Val Gly 992at gac tgg tgg agg aat gag cag ttc tgg gtc att gga ggt gtg 2957 Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val 925 93cc tca cac ctc ttt gct gtg ttc cag gga ctt ctc aag gtc ata gct 3 Ser His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Ile Ala 945tt gat aca agc ttc acc gtg aca tca aag ggt gga gat gat gag 3 Val Asp Thr Ser Phe Thr Val Thr Ser Lys Gly Gly Asp Asp Glu 955 967tc tca gag cta tat aca ttc aaa tgg act acc tta ttg ata cct 3 Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro 975 98ct acc acc ttg ctt cta ttg aac ttc att ggt gtg gtc gct ggc gtt 3 Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala Gly Val 99 aat gcg atc aat aac gga tat gag tca tgg ggc ccc ctc ttt ggg 3 Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly aag cta ttc ttt gca ttt tgg gtg att gtc cat ctt tat ccc ttt ctc 3245 Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu 25 a ggt ttg gtt gga agg caa aac agg aca cca acg att gtc atc gtc 3293 Lys Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val 4g tcc att ctg ctg gct tca atc ttc tcg ctc ctt tgg gtt cgg att 334er Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile 6gat cct ttc ctt gcg aag gat gat ggt ccg ctt ctt gag gag tgt ggt 3389 Asp Pro Phe Leu Ala Lys Asp Asp Gly Pro Leu Leu Glu Glu Cys Gly 75 g gat tgc aac taggatgtca gtgcatcagc tcccccaatc tgcatatgct 344sp Cys Asn aagtatat tttctggtgt ttgtccccat attcagtgtc tgtagataag agacatgaaa 35ccaagt ttcttttgat ccatggtgaa cctacttaat atctgagaga tatactgggg 356tggag gctgcggcaa tccttgtgca gttgggccgt ggaatacagc atatgcaagt 362attgt gcagcattct ttattacttg gtcgcaatat agatgggctg agccgaacag 368tattt tgattctgca ctgctcccgt gtacaaactt ggttctcaat aaggcaggca 374gcatc tgccagtgga acagagcaac ctgcacatta tttatgtatg cctgttcatt 38ggcttg ttcattacat gttcgtctat actagaaaaa acagaatatt agcattaatc 386ttaat taaagtatgt aaatgcgcct gttttttgtt gtgtactgta atcatctgag 392tttgt gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3969 PRT Zea mays Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu Val Val Ile Arg Arg Asp Gly Asp Pro Gly Pro Lys Pro Pro Arg 2 Glu Gln Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Asp Val Gly Leu 35 4a Pro Gly Gly Asp Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro 5 Val Cys Arg Asp Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr Gln Asn 65 7 Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Leu Lys Gly Cys Gln Arg 85 9l Thr Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Asp Asn Glu Asn Trp Asp Gly His Asp Ser Gln Ser Val Ala Glu Ser Met Leu Gly His Met Ser Tyr Gly Arg Gly Gly Asp Pro Asn Gly Ala Pro Ala Phe Gln Leu Asn Pro Asn Val Pro Leu Leu Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His Ala Leu Val Pro Ser Phe Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Tyr Ala Asp Pro Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys Asp Leu Ala 2Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met Glu Asn Trp 222ln Arg Gln Glu Arg Met His Gln Thr Gly Asn Asp Gly Gly Gly 225 234sp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu Ala Arg Gln 245 25ln Leu Ser Arg Lys Ile Pro Leu Pro Ser Ser Gln Ile Asn Pro Tyr 267et Ile Ile Ile Ile Arg Leu Val Val Leu Gly Phe Phe Phe His 275 28yr Arg Val Met His Pro Val Asn Asp Ala Phe Ala Leu Trp Leu Ile 29Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile Leu Asp Gln 33Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg Leu 325 33er Leu Arg Phe Asp Lys Glu Gly Gln Pro Ser Gln Leu Ala Pro Ile 345he Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Val 355 36hr Thr Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp 378al Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe 385 39Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp Val Pro Phe 44Lys Arg Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Gln 423ys Ile Asp Tyr Leu Lys Asp Lys Val Ala Ala Asn Phe Val Arg 435 44lu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile 456la Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr 465 478ln Asp Gly Thr Pro Trp Pro Gly Asn Asn Val Arg Asp His Pro 485 49ly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly Leu Asp Cys Glu 55Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro 5525 Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg 534er Ala Val Leu Thr Asn Ala Pro Tyr Leu Leu Asn Leu Asp Cys 545 556is Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala Met Cys Phe 565 57et Met Asp Pro Leu Leu Gly Lys Lys Val Cys Tyr Val Gln Phe Pro 589rg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ala Asn Arg Asn 595 6Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly 662le Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr 625 634yr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg Thr Cys Asn 645 65ys Trp Pro Lys Trp Cys Phe Cys Cys Cys Cys Phe Gly Asn Arg Lys 667ys Lys Thr Thr Lys Pro Lys Thr Glu Lys Lys Lys Leu Leu Phe 675 68he Lys Lys Glu Glu Asn Gln Ser Pro Ala Tyr Ala Leu Gly Glu Ile 69Glu Ala Ala Pro Gly Ala Glu Asn Glu Lys Ala Gly Ile Val Asn 77Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser Ser Val Phe Val Thr 725 73er Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys Ser Ala Ser Pro Ala 745eu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp 755 76ys Thr Asp Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr 778sp Ile Leu Thr Gly Phe Lys Met His Cys His Gly Trp Arg Ser 785 79Tyr Cys Ile Pro Lys Arg Val Ala Phe Lys Gly Ser Ala Pro Leu 88Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly Ser 823lu Ile Phe Phe Ser Asn His Cys Pro Leu Trp Tyr Gly Tyr Gly 835 84ly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr Ile Asn Ser Ile Val 856ro Trp Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Leu Pro Ala 865 878ys Leu Leu Thr Gly Lys Phe Ile Thr Pro Glu Leu Asn Asn Val 885 89la Ser Leu Trp Phe Met Ser Leu Phe Ile Cys Ile Phe Ala Thr Ser 99Leu Glu Met Arg Trp Ser Gly Val Gly Ile Asp Asp Trp Trp Arg 9925 Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ser His Leu Phe Ala 934he Gln Gly Leu Leu Lys Val Ile Ala Gly Val Asp Thr Ser Phe 945 956al Thr Ser Lys Gly Gly Asp Asp Glu Glu Phe Ser Glu Leu Tyr 965 97hr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu Leu 989sn Phe Ile Gly Val Val Ala Gly Val Ser Asn Ala Ile Asn Asn 995 Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Val Gly Arg 3n Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser Ile Leu Leu Ala 5Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Leu Ala Lys 65 p Asp Gly Pro Leu Leu Glu Glu Cys Gly Leu Asp Cys Asn 8NA Zea mays aggcga gcgccgggct ggtgg 25 2A Zea mays 2tgcaa tccaaaccac actcc
25 2DNA Zea mays CDS ((34gcagcagcag caccaccact gcgcggcatt gcagcgagca agcgggaggg atctggggca 6gcggt cgctgccgct gccgctcgga tctagagggc cgcacgggct gattgccctc cggcctc gtcggtgtcg gtggagtgtg aatcggtgtg tgtaggagga gcgcggag gcg gcc aac aag ggg atg gtg gca ggc tct cac aac cgc aac gag 226 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu gtc atg atc cgc cac gac ggc gac gcg cct gtc ccg gct aag ccc 274 Phe Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 2 acg aag agt gcg aat ggg cag gtc tgc cag att tgt ggc gac act gtt 322 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 4c gtt tca gcc act ggt gat gtc ttt gtt gcc tgc aat gag tgt gcc 37al Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 ttc cct gtc tgc cgc cct tgc tat gag tac gag cgc aag gaa ggg aac 4Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 caa tgc tgc cct cag tgc aag act aga tac aag aga cag aaa ggt agc 466 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9t cga gtt cat ggt gat gat gag gag gaa gat gtt gat gac ctg gac 5Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp gaa ttc aac tat aag caa ggc aat ggg aag ggc cca gag tgg cag 562 Asn Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln caa gga gat gac gct gat ctg tct tca tct gct cgc cat gac cca 6Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro cat cgg att cca cgc ctt aca agt gga caa cag ata tct gga gag 658 His His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu atc cct gat gca tcc cct gac cgt cat tct atc cgc agt cca aca tcg 7Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser tat gtt gat cca agc gtt cca gtt cct gtg agg att gtg gac ccc 754 Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro aag gac ttg aat tcc tat ggg ctt aat agt gtt gac tgg aag gaa 8Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2gtt gag agc tgg agg gtt aaa cag gac aaa aat atg ttg caa gtg 85al Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 222at aaa tat cca gag gct aga gga gac atg gag ggg act ggc tca 898 Thr Asn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser 225 234ga gaa gat atg caa atg gtt gat gat gca cgc cta cct ttg agc 946 Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 25gc att gtg cca att tcc tca aac cag ctc aac ctt tac cgg ata gta 994 Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 267tt ctc cgt ctt atc atc ctg tgc ttc ttc ttc caa tat cgt atc e Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 28gt cat cca gtg cgt aat gct tat gga ttg tgg cta gta tct gtt atc r His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 29gag gtc tgg ttt gcc ttg tcc tgg ctt cta gat cag ttc cca aaa s Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys 33tgg tat cca atc aac cgt gag aca tat ctc gac agg ctt gca ttg agg p Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 33at gat aga gag gga gag cca tca cag ctg gct ccc att gat gtc ttt r Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 345gt aca gtg gat cca ttg aag gaa cct cca ctg atc aca gcc aac l Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 36ct gtt ttg tcc att ctt gct gtg gat tac cct gtt gac aaa gtg tca r Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 378at gtt tct gat gat ggc tca gct atg ctg act ttt gag tct ctc s Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu 385 39gaa act gcc gaa ttt gct aga aag tgg gtt ccc ttt tgt aag aag r Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 44aat att gaa cca aga gct cca gaa ttt tac ttt gct caa aaa ata s Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 423ac ctg aag gac aaa att caa cct tca ttt gtt aag gaa aga cga p Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 44ca atg aag aga gag tat gaa gaa ttc aaa ata aga atc aat gcc ctt a Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 456cc aaa gca cag aaa gtg cct gaa gag ggg tgg acc atg gct gat l Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp 465 478ct gct tgg cct ggg aat aac cct agg gac cat cct ggc atg att y Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 49ag gtg ttc ttg ggg cac agt ggt ggg ctt gac act gat gga aat gaa n Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 55cca cgt ctt gtc tat gtc tct cgt gaa aag aga cca ggc ttt cag u Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln 5525 cat cac aag aag gct ggt gca atg aat gca ctg att cgt gta tct gct s His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 534tg aca aat ggt gcc tat ctt ctc aat gtg gat tgt gac cat tac l Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr 545 556at agc agc aaa gct ctt aga gaa gca atg tgc ttc atg atg gat e Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 57ca gct cta gga agg aaa act tgt tat gta caa ttt cca caa aga ttt o Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 589gc att gac ttg cac gat cga tat gct aat agg aac ata gtc ttc 2 Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 6ttt gat atc aac atg aaa ggt cta gat ggc att cag ggt cca gtc tat 2 Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 662ga aca gga tgc tgt ttc aat agg cag gct ttg tat gga tat gat 2 Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp 625 634tt ttg act gaa gct gat ctg gaa cct aac att gtt gtt aag agc 2 Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 65gc tgt ggt aga agg aag aga aag aac aag agt tat atg gat agt caa 2 Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 667gt att atg aag aga aca gaa tct tca gct ccc atc ttt aac atg 2242 Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 68aa gac atc gag gag ggt att gaa ggt tat gag gat gaa agg tca gtg 229sp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 69atg tcc cag agg aaa ttg gag aaa cgc ttt ggt cag tct cca atc 2338 Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile 77ttc att gca tcc acc ttt atg act caa ggt ggc ata cca cct tca aca 2386 Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 73ac cca gct tct cta ctg aag gaa gct atc cat gtt atc agc tgt ggg 2434 Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 745ag gac aaa act gaa tgg gga aaa gag att ggc tgg atc tat ggt 2482 Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 76ca gtt aca gag gat att ctg act ggg ttt aaa atg cat gca aga ggc 253al Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 778aa tca atc tac tgc atg cca cca cga cct tgt ttc aag ggt tct 2578 Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser 785 79cca atc aat ctt tct gat cgt ctt aat cag gtg ctc cgt tgg gct 2626 Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 88ggg tca gtg gaa att ctg ctt agc aga cat tgt cct ata tgg tat 2674 Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 823ac aat ggg cga ttg aag ctt ttg gag agg ctg gct tac att aac 2722 Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 84cc att gtt tat cca atc aca tct gtt ccg ctt atc gcc tat tgt gtg 277le Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 856ct gct atc tgt ctt ctt acc aat aaa ttt atc att cct gag att 28Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile 865 878at tat gct gga atg ttc ttc att ctt ctt ttt gcc tcc att ttc 2866 Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 89ca act ggt ata ttg gag ctc aga tgg agt ggt gtt ggc att gaa gat 29Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 99tgg aga aat gag cag ttt tgg gtt att ggt ggc acc tct gcc cat 2962 Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 9925 ctc ttc gcg gtg ttc cag ggt ctg ctg aaa gtg ttg gct ggg att gat 3 Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 934ac ttc aca gtt acc tca aag gca tct gat gag gat ggc gac ttt 3 Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 945 956ag cta tat gtg ttc aag tgg acc agt ttg ctc atc cct ccg acc 3 Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 97ct gtt ctt gtc att aac ctg gtc gga atg gtg gca gga att tcg tat 3 Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 989tt aac agc ggc tac caa tcc tgg ggt ccg ctc ttt gga aag ctg 32Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 ttc tcg atc tgg gtg atc ctc cat ctc tac ccc ttc ctc aag ggt 325he Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly ctc atg ggc agg cag aac cgc acg cca aca atc gtc atc gtt tgg tcc 3298 Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser 3c ctc ctt gcg tct atc ttc tcc ttg ctg tgg gtg aag atc gat cct 3346 Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 5ttc atc tcc ccg aca cag aaa gct gcc gcc ttg ggg caa tgt ggt gtg 3394 Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 65 c tgc tgatccagat tgtgactctt atctgaagag gctcagccaa agatctgccc 345ys cctcgtgtaa atacctgagg gggctagatg ggaatttttt gttgtagatg aggatggatc 35tccaag ttatgcctct gtttattagc ttcttcggtg ccggtgctgc tgcagacaat 357agcct ttctaccttg cttgtagtgc tggccagcag cgtaaattgt gaattctgca 363ttata cgtggtgttt attgttttag agtaaattat catttgtttg aggtaactat 369cgaac tatatggcaa tgctgttatt taaaa 3725 22 T Zea mays 22 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 2 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 4y Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9o Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 222sn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser 225 234ly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 25rg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 267le Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 28er His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 29Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys 33Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 33yr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 345er Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 36hr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 378yr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu 385 39Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 44Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 423yr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 44la Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 456la Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp 465 478hr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 49ln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 55Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln 5525 His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 534eu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr 545 556sn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 57ro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 589ly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 6Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 6
62ly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp 625 634al Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 65ys Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 667rg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 68lu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 69Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile 77Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 73sn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 745lu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 76er Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 778ln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser 785 79Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 88Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 823yr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 84hr Ile Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 856ro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile 865 878sn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 89la Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 99Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 9925 Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 934sn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 945 956lu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 97hr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 989le Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser 3e Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 5Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 65 n Cys 23 25 DNA Zea mays 23 atggcggcca acaaggggat ggtgg 25 24 25 DNA Zea mays 24 tcagcagttc acaccacatt gcccc 25 25 38Zea mays CDS (23496) 25 ccacagctca tataccaaga gccggagcag cttagcgcag cccagagcgg cgccgcgcca 6aaccc ccacccgcca cagccgcgtg cgcatgtgag cggtcgccgc ggccgggaga gaggagg ggaggactac gtgcatttcg ctgtgccgcc gccgcggggt tcgtgcgcga agatccg gcggggcggg gcggggggcc tgag atg gag gct agc gcg ggg ctg 235 Met Glu Ala Ser Ala Gly Leu gcc ggc tcg cat aac cgg aac gag ctg gtg gtg atc cgc cgc gac 283 Val Ala Gly Ser His Asn Arg Asn Glu Leu Val Val Ile Arg Arg Asp ag tcg gga gcc gcg ggc ggc ggc gcg gcg cgc cgg gcg gag gcg 33lu Ser Gly Ala Ala Gly Gly Gly Ala Ala Arg Arg Ala Glu Ala 25 3g tgc cag ata tgc ggc gac gag gtc ggg gtg ggc ttc gac ggg gag 379 Pro Cys Gln Ile Cys Gly Asp Glu Val Gly Val Gly Phe Asp Gly Glu 4 55 ccc ttc gtg gcg tgc aac gag tgc gcc ttc ccc gtc tgc cgc gcc tgc 427 Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro Val Cys Arg Ala Cys 6 tac gag tac gag cgc cgc gag ggc tcg caa gcg tgc ccg cag tgc agg 475 Tyr Glu Tyr Glu Arg Arg Glu Gly Ser Gln Ala Cys Pro Gln Cys Arg 75 8c cgc tac aag cgc ctc aag ggc tgc ccg cgg gtg gcc ggc gac gag 523 Thr Arg Tyr Lys Arg Leu Lys Gly Cys Pro Arg Val Ala Gly Asp Glu 9ag gac ggc gtc gac gac ctg gag ggc gag ttc ggc ctg cag gac 57lu Asp Gly Val Asp Asp Leu Glu Gly Glu Phe Gly Leu Gln Asp gcc gcc cac gag gac gac ccg cag tac gtc gcc gag tcc atg ctc 6Ala Ala His Glu Asp Asp Pro Gln Tyr Val Ala Glu Ser Met Leu agg gcg cag atg agc tac ggc cgc ggc ggc gac gcg cac ccc ggc ttc 667 Arg Ala Gln Met Ser Tyr Gly Arg Gly Gly Asp Ala His Pro Gly Phe ccc gtc ccc aac gtg ccg ctc ctc acc aac ggc cag atg gtt gat 7Pro Val Pro Asn Val Pro Leu Leu Thr Asn Gly Gln Met Val Asp atc ccg ccg gag cag cac gcg ctc gtg ccg tcc tac atg agc ggc 763 Asp Ile Pro Pro Glu Gln His Ala Leu Val Pro Ser Tyr Met Ser Gly ggc ggc ggg ggc aag agg atc cac ccg ctc cct ttc gca gat ccc 8Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Phe Ala Asp Pro ctt cca gtg caa ccg aga tcc atg gac ccg tcc aag gat ctg gcc 859 Asn Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys Asp Leu Ala 22gcc tac gga tat ggc agc gtg gcc tgg aag gag aga atg gag ggc tgg 9Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met Glu Gly Trp 223ag aag cag gag cgc ctg cag cat gtc agg agc gag ggt ggc ggt 955 Lys Gln Lys Gln Glu Arg Leu Gln His Val Arg Ser Glu Gly Gly Gly 235 24at tgg gat ggc gac gat gca gat ctg cca cta atg gat gaa gct agg p Trp Asp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu Ala Arg 256ca ttg tcc aga aaa gtc cct ata tca tca agc cga att aat ccc n Pro Leu Ser Arg Lys Val Pro Ile Ser Ser Ser Arg Ile Asn Pro 265 27ac agg atg att atc gtt atc cgg ttg gtg gtt ttg ggt ttc ttc ttc r Arg Met Ile Ile Val Ile Arg Leu Val Val Leu Gly Phe Phe Phe 289ac tac cga gtg atg cat ccg gcg aaa gat gca ttt gca ttg tgg ctc s Tyr Arg Val Met His Pro Ala Lys Asp Ala Phe Ala Leu Trp Leu 33tct gta atc tgt gaa atc tgg ttt gcg atg tcc tgg att ctt gat e Ser Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile Leu Asp 3325 cag ttc cca aag tgg ctt cca atc gag aga gag act tac ctg gac cgt n Phe Pro Lys Trp Leu Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg 334ca cta agg ttt gac aag gaa ggt caa ccc tct cag ctt gct cca u Ser Leu Arg Phe Asp Lys Glu Gly Gln Pro Ser Gln Leu Ala Pro 345 35tc gac ttc ttt gtc agt acg gtt gat ccc aca aag gaa cct ccc ttg e Asp Phe Phe Val Ser Thr Val Asp Pro Thr Lys Glu Pro Pro Leu 367tc aca gcg aac act gtc ctt tcc atc ctt tct gtg gat tat ccg gtt l Thr Ala Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val 389ag gtc tcc tgc tat gtt tct gat gat ggt gct gca atg ctt acg u Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr 395 4ttt gaa gca ttg tct gaa aca tct gaa ttt gca aag aaa tgg gtt cct e Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp Val Pro 442gc aaa aag ttt aat atc gag cct cgt gct cct gag tgg tac ttc e Ser Lys Lys Phe Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe 425 43aa cag aag ata gac tac ctg aaa gac aag gtt gct gct tca ttt gtt n Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Ala Ala Ser Phe Val 445gg gag agg agg gcg atg aag aga gaa tac gag gaa ttc aag gta agg g Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg 467at gcc ttg gtt gca aaa gcc caa aag gtt cct gag gaa gga tgg e Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp 475 48ca atg caa gat gga agc ccc tgg cct gga aac aac gta cgc gat cat r Met Gln Asp Gly Ser Pro Trp Pro Gly Asn Asn Val Arg Asp His 49gga atg att cag gta ttc ctt ggc caa agt ggc ggt cgt gat gtg o Gly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly Arg Asp Val 55gga aat gag ttg cct cgc ctg gtt tat gtc tcg aga gaa aag agg u Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 523ca ggt tat aac cat cac aag aag gct ggt gcc atg aat gca ctg gtc o Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val 545tc tct gct gtc tta tca aat gct gca tac cta ttg aac ttg gac g Val Ser Ala Val Leu Ser Asn Ala Ala Tyr Leu Leu Asn Leu Asp 555 56gt gat cac tac atc aac aat agc aag gcc ata aaa gag gct atg tgt s Asp His Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala Met Cys 578tg atg gat cct ttg gtg ggg aag aaa gtg tgc tat gta cag ttc 2 Met Met Asp Pro Leu Val Gly Lys Lys Val Cys Tyr Val Gln Phe 585 59ct cag agg ttt gat ggt att gac aaa aat gat cga tac gct aac agg 2 Gln Arg Phe Asp Gly Ile Asp Lys Asn Asp Arg Tyr Ala Asn Arg 66aac gtt gtc ttt ttt gac atc aac atg aaa ggt ttg gac ggt att caa 2 Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln 623cc att tat gtg ggt act gga tgt gtt ttc aga cgg cag gca ctg 2 Pro Ile Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln Ala Leu 635 64at ggt tat gat gct cct aaa acg aag aag cca cca tca aga act tgc 22Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg Thr Cys 656gc tgg ccc aag tgg tgc ctc tct tgc tgc tgc agc agg aac aag 225ys Trp Pro Lys Trp Cys Leu Ser Cys Cys Cys Ser Arg Asn Lys 665 67at aaa aag aag act aca aaa cca aag acg gag aag aag aaa aga tta 2299 Asn Lys Lys Lys Thr Thr Lys Pro Lys Thr Glu Lys Lys Lys Arg Leu 689tt ttc aag aaa gca gaa aac cca tct cct gca tat gct ttg ggt gaa 2347 Phe Phe Lys Lys Ala Glu Asn Pro Ser Pro Ala Tyr Ala Leu Gly Glu 77gat gaa ggt gct cca ggt gct gat atc gag aag gcc gga atc gta 2395 Ile Asp Glu Gly Ala Pro Gly Ala Asp Ile Glu Lys Ala Gly Ile Val 7725 aat caa cag aaa cta gag aag aaa ttt ggg cag tct tct gtt ttt gtc 2443 Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser Ser Val Phe Val 734ca aca ctt ctt gag aac gga ggg acc ctg aag agc gca agt cca 249er Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys Ser Ala Ser Pro 745 75ct tct ctt ctg aag gaa gct ata cat gtt atc agc tgc ggc tac gaa 2539 Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu 767ac aag acc gac tgg gga aaa gag att ggc tgg att tac gga tcg atc 2587 Asp Lys Thr Asp Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Ile 789ag gat atc ttg act gga ttt aag atg cac tgc cat ggc tgg cgg 2635 Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys His Gly Trp Arg 795 8tct att tac tgc atc ccg aag cgg cct gca ttc aaa ggt tct gcg cct 2683 Ser Ile Tyr Cys Ile Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro 882ac ctt tcc gac cgt ctt cac cag gtc ctt cgc tgg gcc ctt ggg 273sn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly 825 83cc gtc gaa att ttc ttc agc aag cac tgc cca ctt tgg tac gga tac 2779 Ser Val Glu Ile Phe Phe Ser Lys His Cys Pro Leu Trp Tyr Gly Tyr 845gc ggc ggg cta aaa ttc ctg gaa agg ttt tct tat atc aac tcc atc 2827 Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr Ile Asn Ser Ile 867at ccc tgg acg tcc att cct ctc ctg gct tac tgt acc ttg cct 2875 Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Leu Pro 875 88cc atc tgc ctg ctc acg ggg aag ttt atc aca cca gag ctt acc aat 2923 Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr Pro Glu Leu Thr Asn 89gcc agt atc tgg ttc atg gca ctt ttc atc tgc atc tcc gtg acc 297la Ser Ile Trp Phe Met Ala Leu Phe Ile Cys Ile Ser Val Thr 99atc ctg gaa atg agg tgg agt ggc gtg gcc atc gac gac tgg tgg 3 Ile Leu Glu Met Arg Trp Ser Gly Val Ala Ile Asp Asp Trp Trp 923gg aac gag cag ttc tgg gtc atc gga ggc gtt tcg gcg cat ctg ttc 3 Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe 945tg ttc cag ggc ctg ctg aag gtg ttc gcc ggc atc gac acg agc 3 Val Phe Gln Gly Leu Leu Lys Val Phe Ala Gly Ile Asp Thr Ser 955 96tc acc gtg acg tcg aag gcc ggg gac gac gag gag ttc tcg gag ctg 3 Thr Val Thr Ser Lys Ala Gly Asp Asp Glu Glu Phe Ser Glu Leu 978cg ttc aag tgg acc acc ctg ctg ata ccc ccg acc acg ctc ctc 32Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu 985 99tg ctg aac ttc atc ggg gtg gtg gcc ggg atc tcg aac gcg atc aac 3259 Leu Leu Asn Phe Ile Gly Val Val Ala Gly Ile Ser Asn Ala Ile Asn c ggg tac gag tcg tgg ggc ccc ctg ttc ggg aag ctc ttc ttc gcc 33Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala 25 c tgg gtg atc gtc cac ctg tac ccg ttc ctc aag ggt ctg gtg ggg 3355 Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Val Gly 4agg cag aac agg acg ccg acg atc gtc atc gtc tgg tcc atc ctg ctg 34Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser Ile Leu Leu 55 c tcg atc ttc tcg ctc ctg tgg gtc cgc gtc gac ccg ttc ctc gcc 345er Ile Phe Ser Leu Leu Trp Val Arg Val Asp Pro Phe Leu Ala 7aag agc aac ggc ccg ctc ctg gag gag tgt ggc ctg gac tgc aac 3496 Lys Ser Asn Gly Pro Leu Leu Glu Glu Cys Gly Leu Asp Cys Asn 85 aagtgggg gccccctgtc actcgaagtt ctgtcacggg cgaattacgc ctgatttttt 3556 gttgttgttg ttgttggaat tctttgctgt agatagaaac cacatgtcca cggcatctct 36tgtcca ttggagcagg agagaggtgc ctgctgctgt ttgttgagta aattaaaagt 3676 tttaaagtta tacagtgatg cacattccag tgcccagtgt attccctttt tacagtctgt 3736 atattagcga caaaggacat attggttagg agtttgattc ttttgtaaaa aaaaaaaaaa 3796 aaaaaaaaaa aaaaaaa 38 Zea mays 26 Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu Val Val Ile Arg Arg Asp Arg Glu Ser Gly Ala Ala Gly Gly Gly 2 Ala Ala Arg Arg Ala Glu Ala Pro Cys Gln Ile Cys Gly Asp Glu Val 35 4y Val Gly Phe Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Ala Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Ser 65 7 Gln Ala Cys Pro Gln Cys Arg Thr Arg Tyr Lys Arg Leu Lys Gly Cys 85 9o Arg
Val Ala Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Glu Glu Phe Gly Leu Gln Asp Gly Ala Ala His Glu Asp Asp Pro Gln Val Ala Glu Ser Met Leu Arg Ala Gln Met Ser Tyr Gly Arg Gly Asp Ala His Pro Gly Phe Ser Pro Val Pro Asn Val Pro Leu Leu Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His Ala Leu Pro Ser Tyr Met Ser Gly Gly Gly Gly Gly Gly Lys Arg Ile His Leu Pro Phe Ala Asp Pro Asn Leu Pro Val Gln Pro Arg Ser Met 2Pro Ser Lys Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp 222lu Arg Met Glu Gly Trp Lys Gln Lys Gln Glu Arg Leu Gln His 225 234rg Ser Glu Gly Gly Gly Asp Trp Asp Gly Asp Asp Ala Asp Leu 245 25ro Leu Met Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys Val Pro Ile 267er Ser Arg Ile Asn Pro Tyr Arg Met Ile Ile Val Ile Arg Leu 275 28al Val Leu Gly Phe Phe Phe His Tyr Arg Val Met His Pro Ala Lys 29Ala Phe Ala Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe 33Ala Met Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Leu Pro Ile Glu 325 33rg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly 345ro Ser Gln Leu Ala Pro Ile Asp Phe Phe Val Ser Thr Val Asp 355 36ro Thr Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile 378er Val Asp Tyr Pro Val Glu Lys Val Ser Cys Tyr Val Ser Asp 385 39Gly Ala Ala Met Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu 44Ala Lys Lys Trp Val Pro Phe Ser Lys Lys Phe Asn Ile Glu Pro 423la Pro Glu Trp Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp 435 44ys Val Ala Ala Ser Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu 456lu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln 465 478al Pro Glu Glu Gly Trp Thr Met Gln Asp Gly Ser Pro Trp Pro 485 49ly Asn Asn Val Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly 55Ser Gly Gly Arg Asp Val Glu Gly Asn Glu Leu Pro Arg Leu Val 5525 Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala 534la Met Asn Ala Leu Val Arg Val Ser Ala Val Leu Ser Asn Ala 545 556yr Leu Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys 565 57la Ile Lys Glu Ala Met Cys Phe Met Met Asp Pro Leu Val Gly Lys 589al Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Lys 595 6Asn Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met 662ly Leu Asp Gly Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys 625 634he Arg Arg Gln Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys 645 65ys Pro Pro Ser Arg Thr Cys Asn Cys Trp Pro Lys Trp Cys Leu Ser 667ys Cys Ser Arg Asn Lys Asn Lys Lys Lys Thr Thr Lys Pro Lys 675 68hr Glu Lys Lys Lys Arg Leu Phe Phe Lys Lys Ala Glu Asn Pro Ser 69Ala Tyr Ala Leu Gly Glu Ile Asp Glu Gly Ala Pro Gly Ala Asp 77Ile Glu Lys Ala Gly Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe 725 73ly Gln Ser Ser Val Phe Val Ala Ser Thr Leu Leu Glu Asn Gly Gly 745eu Lys Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His 755 76al Ile Ser Cys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Lys Glu Ile 778rp Ile Tyr Gly Ser Ile Thr Glu Asp Ile Leu Thr Gly Phe Lys 785 79His Cys His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Pro 88Phe Lys Gly Ser Ala Pro Leu Asn Leu Ser Asp Arg Leu His Gln 823eu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Phe Ser Lys His 835 84ys Pro Leu Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg 856er Tyr Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu 865 878la Tyr Cys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe 885 89le Thr Pro Glu Leu Thr Asn Val Ala Ser Ile Trp Phe Met Ala Leu 99Ile Cys Ile Ser Val Thr Gly Ile Leu Glu Met Arg Trp Ser Gly 9925 Val Ala Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly 934al Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val 945 956la Gly Ile Asp Thr Ser Phe Thr Val Thr Ser Lys Ala Gly Asp 965 97sp Glu Glu Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu 989ro Pro Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala 995 Ile Ser Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro 3e Leu Lys Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val 5Ile Val Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val 65 g Val Asp Pro Phe Leu Ala Lys Ser Asn Gly Pro Leu Leu Glu Glu 8Cys Gly Leu Asp Cys Asn 25 DNA Zea mays 27 atggaggcta gcgcggggct ggtgg 25 28 25 DNA Zea mays 28 tcagttgcag tccaggccac actcc 25 29 3746 DNA Zea mays CDS (3255taggatcaa aaccgtctcg ccgctgcaat aatcttttgt caattcttaa tccctcgcgt 6gcgac agcggaacca actcacgttg ccgcggcttc ctccatcggt gcggtgccct cttttct ctcgtccctc ctccccccgt atagttaagc cccgccccgc tactactact agcagca gcagcgctct cgcagcggga gatgcggtgt tgatccgtgc cccgctcgga 24ggact ggtgccggct ctgcccaggc cccaggctcc aggccagctc cctcgacgtt 3ggcgag ctcgcttgcc atg gag ggc gac gcg gac ggc gtg aag tcg ggg 353 Met Glu Gly Asp Ala Asp Gly Val Lys Ser Gly agg cgc ggt ggc gga cag gtg tgc cag atc tgc ggc gac ggc gtg ggc 4Arg Gly Gly Gly Gln Val Cys Gln Ile Cys Gly Asp Gly Val Gly 5 acc acg gcg gag ggg gac gtc ttc gcc gcc tgc gac gtc tgc ggg ttt 449 Thr Thr Ala Glu Gly Asp Val Phe Ala Ala Cys Asp Val Cys Gly Phe 3 ccg gtg tgc cgc ccc tgc tac gag tac gag cgc aag gac ggc acg cag 497 Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln 45 5g tgc ccc cag tgc aag acc aag tac aag cgc cac aag ggg agc ccg 545 Ala Cys Pro Gln Cys Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro 6 75 gcg atc cgt ggg gag gaa gga gac gac act gat gcc gat agc gac ttc 593 Ala Ile Arg Gly Glu Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe 8 aat tac ctt gca tct ggc aat gag gac cag aag cag aag att gcc gac 64yr Leu Ala Ser Gly Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp 95 aga atg cgc agc tgg cgc atg aac gtt ggg ggc agc ggg gat gtt ggt 689 Arg Met Arg Ser Trp Arg Met Asn Val Gly Gly Ser Gly Asp Val Gly ccc aag tat gac agt ggc gag atc ggg ctt acc aag tat gac agt 737 Arg Pro Lys Tyr Asp Ser Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser gag att cct cgg gga tac atc cca tca gtc act aac agc cag atc 785 Gly Glu Ile Pro Arg Gly Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile tca gga gaa atc cct ggt gct tcc cct gac cat cat atg atg tcc cca 833 Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His His Met Met Ser Pro ggg aac att ggc aag cgt gct cca ttt ccc tat gtg aac cat tcg 88ly Asn Ile Gly Lys Arg Ala Pro Phe Pro Tyr Val Asn His Ser aat ccg tca agg gag ttc tct ggt agc att ggg aat gtt gcc tgg 929 Pro Asn Pro Ser Arg Glu Phe Ser Gly Ser Ile Gly Asn Val Ala Trp 2gag agg gtt gat ggc tgg aaa atg aag cag gac aag ggg acg att 977 Lys Glu Arg Val Asp Gly Trp Lys Met Lys Gln Asp Lys Gly Thr Ile 22atg acg aat ggc aca agc att gct ccc tct gag ggt cgg ggt gtt o Met Thr Asn Gly Thr Ser Ile Ala Pro Ser Glu Gly Arg Gly Val 223gt gat att gat gca tca act gat tac aac atg gaa gat gcc tta ttg y Asp Ile Asp Ala Ser Thr Asp Tyr Asn Met Glu Asp Ala Leu Leu 245ac gaa act cga cag cct cta tct agg aaa gtt cca ctt cct tcc n Asp Glu Thr Arg Gln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser 255 26cc agg ata aat cca tac agg atg gtc att gtg ctg cga ttg att gtt r Arg Ile Asn Pro Tyr Arg Met Val Ile Val Leu Arg Leu Ile Val 278gc atc ttc ttg cac tac cgt atc aca aat cct gtg cgc aat gca u Ser Ile Phe Leu His Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala 285 29ac cca tta tgg ctt cta tct gtt ata tgt gag atc tgg ttt gct ctt r Pro Leu Trp Leu Leu Ser Val Ile Cys Glu Ile Trp Phe Ala Leu 33tcg tgg ata ttg gat cag ttc cct aag tgg ttt cca atc aac cgg gag r Trp Ile Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu 323ac ctt gat agg ctg gca tta agg tat gac cgg gaa ggt gag cca r Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro 335 34ct cag ttg gct gct gtt gac att ttc gtc agt aca gtc gac cca atg r Gln Leu Ala Ala Val Asp Ile Phe Val Ser Thr Val Asp Pro Met 356ag cct cct ctt gtc act gcc aat acc gtg cta tcc att ctt gct s Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ala 365 37tg gat tac cct gtg gat aag gtc tct tgc tat gta tct gat gat gga l Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly 389ct gcg atg ctg aca ttt gat gca cta gct gag act tca gag ttt gct a Ala Met Leu Thr Phe Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala 44aaa tgg gta cca ttt gtt aag aag tac aac att gaa cct aga gct g Lys Trp Val Pro Phe Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala 4425 cct gaa tgg tac ttc tcc cag aaa att gat tac ttg aag gac aaa gtg o Glu Trp Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val 434ct tca ttt gtt aaa gac cgc cgg gcc atg aag aga gaa tat gaa s Pro Ser Phe Val Lys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu 445 45aa ttc aaa gtt agg gta aat ggc ctt gtt gct aag gca cag aaa gtt u Phe Lys Val Arg Val Asn Gly Leu Val Ala Lys Ala Gln Lys Val 467ct gag gaa gga tgg atc atg caa gat ggc aca cca tgg cca gga aac o Glu Glu Gly Trp Ile Met Gln Asp Gly Thr Pro Trp Pro Gly Asn 489cc agg gac cat cct gga atg att cag gtt ttc ctt ggt cac agt n Thr Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly His Ser 495 5ggt ggc ctt gat act gag ggc aat gag cta ccc cgt ttg gtc tat gtt y Gly Leu Asp Thr Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val 552gt gaa aag cgt cct gga ttc cag cat cac aag aaa gct ggt gcc r Arg Glu Lys Arg Pro Gly Phe Gln His His Lys Lys Ala Gly Ala 525 53tg aat gct ctt gtt cgt gtc tca gct gtg ctt acc aat gga caa tac t Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr 545tg ttg aat ctt gat tgt gat cac tac att aac aac agt aag gct ctc 2 Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Leu 567aa gct atg tgc ttc ctt atg gac cct aac cta gga agg agt gtc 2 Glu Ala Met Cys Phe Leu Met Asp Pro Asn Leu Gly Arg Ser Val 575 58gc tac gtc cag ttt ccc cag aga ttc gat ggc att gac agg aat gat 2 Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg Asn Asp 59tat gcc aac agg aac acc gtg ttt ttc gat att aac ttg aga ggt 2 Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly 66gat ggc atc caa gga cca gtt tat gtc gga act ggc tgt gtt ttc 2225 Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Val Phe 623ac cga aca gct cta tat ggt tat gag ccc cca att aag cag aag aag 2273 Asn Arg Thr Ala Leu Tyr Gly Tyr Glu Pro Pro Ile Lys Gln Lys Lys 645gt ttc ttg tca tca cta tgt ggc ggt agg aag aag gca agc aaa 232ly Phe Leu Ser Ser Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys 655 66ca aag aag ggc tcg gac aag aag aag tcg cag aag cat gtg gac agt 2369 Ser Lys Lys Gly Ser Asp Lys Lys Lys Ser Gln Lys His Val Asp Ser 678tg cca gta ttc aac ctt gaa gat ata gag gag gga gtt gaa ggc 24Val Pro Val Phe Asn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly 685 69ct gga ttt gac gac gag aaa tca ctt ctt atg tct caa atg agc ctg 2465 Ala Gly Phe Asp Asp Glu Lys Ser Leu Leu Met Ser Gln Met Ser Leu 77gag aag aga ttt ggc cag tcc gca gcg ttt gtt gcc tcc act ctg atg 25Lys Arg Phe Gly Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met 723at ggt ggt gtt cct cag tcc gca act ccg gag tct ctt ctg aaa 256yr Gly Gly Val Pro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys 735 74aa gct atc cat gtt ata agc tgt ggc tat gag gac aag act gaa tgg 26Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp 756ct gag atc ggg tgg atc tac ggt tct gtg aca gaa gac att ctc 2657 Gly Thr Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu 765 77cc gga ttc aag atg cac gcg cga ggc tgg cgg tcg atc tac tgc atg 27Gly Phe Lys Met His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met 789cc aag cgg cca gct ttc aag ggg tct gcc ccc atc aat ctt tcg gac 2753 Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp 88ctg aac cag gtg ctc cgg tgg gct ctt ggg tcc gtg gag atc ctc 28Leu Asn Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu 8825 ttc agc cgg cac tgc ccc ctg tgg tac ggc tac gga ggg cgg ctc aag 2849 Phe Ser Arg His Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys 834tg gag aga ttc gcg tac atc aac acc acc atc tac ccg ctc
acg 2897 Phe Leu Glu Arg Phe Ala Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr 845 85cc atc ccg ctt ctc atc tac tgc atc ctg ccc gcc atc tgt ctg ctc 2945 Ser Ile Pro Leu Leu Ile Tyr Cys Ile Leu Pro Ala Ile Cys Leu Leu 867cc gga aag ttc atc att cca gag atc agc aac ttc gcc agc atc tgg 2993 Thr Gly Lys Phe Ile Ile Pro Glu Ile Ser Asn Phe Ala Ser Ile Trp 889tc tcc ctc ttc atc tcg atc ttc gcc acg ggc atc ctg gag atg 3 Ile Ser Leu Phe Ile Ser Ile Phe Ala Thr Gly Ile Leu Glu Met 895 9agg tgg agc ggg gtg ggc atc gac gag tgg tgg agg aac gag cag ttc 3 Trp Ser Gly Val Gly Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe 992tg atc ggg ggc atc tcc gcg cac ctc ttc gcc gtg ttc cag ggc 3 Val Ile Gly Gly Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly 925 93tg ctc aag gtg ctg gcc ggc atc gac acc aac ttc acc gtc acc tcc 3 Leu Lys Val Leu Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser 945ag gcc tcg gac gag gac ggc gac ttc gcg gag ctg tac atg ttc aag 3233 Lys Ala Ser Asp Glu Asp Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys 967cg acg ctc ctg atc ccg ccc acc acc atc ctg atc atc aac ctg 328hr Thr Leu Leu Ile Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu 975 98tc ggc gtc gtc gcc ggc atc tcc tac gcc atc aac agc gga tac cag 3329 Val Gly Val Val Ala Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln 99 tgg ggc ccg ctc ttc ggc aag ctc ttc ttc gcc ttc tgg gtc atc 3377 Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile gtc cac ctg tac ccg ttc ctc aag ggc ctc atg ggc agg cag aac cgc 3425 Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg 25 35 acc ccg acc atc gtc gtc gtc tgg gcc atc ctg ctg gcg tcc atc ttc 3473 Thr Pro Thr Ile Val Val Val Trp Ala Ile Leu Leu Ala Ser Ile Phe 45 c ttg ctg tgg gtt cgc atc gac ccc ttc acc acc cgc gtc act ggc 352eu Leu Trp Val Arg Ile Asp Pro Phe Thr Thr Arg Val Thr Gly 6ccg gat acc cag acg tgt ggc atc aac tgc tagggaagtg gaaggtttgt 357sp Thr Gln Thr Cys Gly Ile Asn Cys 75 actttgtaga aacggaggaa taccacgtgc catctgttgt ctgttaagtt atatatatat 363gcaag tggcgttatt tacagctacg tacagaccag tggatattgt ttaccacaaa 369acttg tgttaatatg cattcttttg ttgatataaa aaaaaaaaaa aaaaa 3746 3PRT Zea mays 3lu Gly Asp Ala Asp Gly Val Lys Ser Gly Arg Arg Gly Gly Gly Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly 2 Asp Val Phe Ala Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro 35 4s Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys 5 Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Ala Ile Arg Gly Glu 65 7 Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe Asn Tyr Leu Ala Ser 85 9y Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp Arg Met Arg Ser Trp Met Asn Val Gly Gly Ser Gly Asp Val Gly Arg Pro Lys Tyr Asp Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser Gly Glu Ile Pro Arg Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His His Met Met Ser Pro Thr Gly Asn Ile Gly Arg Ala Pro Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg Phe Ser Gly Ser Ile Gly Asn Val Ala Trp Lys Glu Arg Val Asp 2Trp Lys Met Lys Gln Asp Lys Gly Thr Ile Pro Met Thr Asn Gly 222er Ile Ala Pro Ser Glu Gly Arg Gly Val Gly Asp Ile Asp Ala 225 234hr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg 245 25ln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser Ser Arg Ile Asn Pro 267rg Met Val Ile Val Leu Arg Leu Ile Val Leu Ser Ile Phe Leu 275 28is Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu Trp Leu 29Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp 33Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg 325 33eu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Ala 345sp Ile Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro Leu 355 36al Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val 378ys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr 385 39Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp Val Pro 44Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe 423ln Lys Ile Asp Tyr Leu Lys Asp Lys Val His Pro Ser Phe Val 435 44ys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg 456sn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp 465 478et Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His 485 49ro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr 55Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 5525 Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val 534al Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp 545 556sp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys 565 57he Leu Met Asp Pro Asn Leu Gly Arg Ser Val Cys Tyr Val Gln Phe 589ln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg 595 6Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln 662ro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu 625 634ly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe Leu Ser 645 65er Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys Ser Lys Lys Gly Ser 667ys Lys Lys Ser Gln Lys His Val Asp Ser Ser Val Pro Val Phe 675 68sn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe Asp Asp 69Lys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg Phe Gly 77Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val 725 73ro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val 745er Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly 755 76rp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 778la Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala 785 79Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val 88Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Phe Ser Arg His Cys 823eu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe 835 84la Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu 856yr Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile 865 878ro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser Leu Phe 885 89le Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val 99Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly 9925 Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu 934ly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu 945 956ly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu 965 97le Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala 989le Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu 995 Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val 3l Val Trp Ala Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val 5Arg Ile Asp Pro Phe Thr Thr Arg Val Thr Gly Pro Asp Thr Gln Thr 65 s Gly Ile Asn Cys 25 DNA Zea mays 3gggcg acgcggacgg cgtga 25 32 25 DNA Zea mays 32 ctagcagttg atgccacacg tctgg 25 33 3753 DNA Zea mays CDS ((34cagcagcaga agcactgcgc ggcattgcag cgatcgagcg ggaggaattt ggggcatggt 6ccaac gccgctcgga tctagaggcc cgcacgggcc gattggtctc cgcccgcctc ggtgttg gtgtcgttgg cgtgtggagc cgtctcggtg ggagcagcgg ggagggagcg atg gcg gcc aac aag ggg atg gtg gcg ggc tcg cac aac cgc aac 228 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn ttc gtc atg atc cgc cac gac ggc gat gtg ccg ggc tcg gct aag 276 Glu Phe Val Met Ile Arg His Asp Gly Asp Val Pro Gly Ser Ala Lys 2 ccc aca aag agt gcg aat gga cag gtc tgc cag att tgc ggt gac tct 324 Pro Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Ser 35 4g ggt gtt tca gcc act ggt gat gtc ttt gtt gcc tgc aat gag tgt 372 Val Gly Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys 5 gcc ttc cct gtc tgc cgc cca tgc tat gag tat gag cgc aag gag ggg 42he Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly 65 7c caa tgc tgc ccc cag tgc aag act aga tac aag aga cag aaa ggt 468 Asn Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly 8 95 agc cct cga gtt cat ggt gat gag gat gag gaa gat gtt gat gac cta 5Pro Arg Val His Gly Asp Glu Asp Glu Glu Asp Val Asp Asp Leu aat gaa ttc aac tac aag caa ggc agt ggg aaa ggc cca gag tgg 564 Asp Asn Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys Gly Pro Glu Trp ctg caa gga gat gat gct gat ctg tct tca tct gct cgc cat gag 6Leu Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Glu cat cat cgg att cca cgc ctg aca agc ggt caa cag ata tct gga 66is His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly att cct gat gct tcc cct gac cgt cat tct atc cgc agt cca aca 7Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr tcg agc tat gtt gat cca agc gtc cca gtt cct gtg agg att gtg gac 756 Ser Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp tcg aag gac ttg aat tcc tat ggg ctt aat agt gtt gac tgg aag 8Ser Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys 2aga gtt gag agc tgg agg gtt aaa cag gac aaa aat atg atg caa 852 Glu Arg Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Met Gln 222ct aat aaa tat cca gag gct aga gga gga gac atg gag ggg act 9Thr Asn Lys Tyr Pro Glu Ala Arg Gly Gly Asp Met Glu Gly Thr 225 23gc tca aat gga gaa gat atg caa atg gtt gat gat gca cgg cta cct 948 Gly Ser Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro 245tg agc cgt atc gtg cca att tcc tca aac cag ctc aac ctt tac cgg 996 Leu Ser Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg 267tg atc att ctc cgt ctt atc atc ctg tgc ttc ttc ttc cag tat l Val Ile Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr 275 28gt gtc agt cat cca gtg cgt gat gct tat gga tta tgg cta gta tct g Val Ser His Pro Val Arg Asp Ala Tyr Gly Leu Trp Leu Val Ser 29atc tgc gag gtc tgg ttt gcc ttg tct tgg ctt cta gat cag ttc l Ile Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe 33aaa tgg tat cca atc aac cgt gag aca tat ctt gac agg ctt gca o Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala 323tg agg tat gat aga gag gga gag cca tca cag ctg gct ccc att gat u Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp 345tc gtc agt aca gtg gat cca ttg aag gaa cct cca ctg atc aca l Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr 355 36cc aac act gtt ttg tcc att ctt tct gtg gat tac cct gtt gac aaa a Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp Lys 378ca tgc tat gtt tct gat gat ggt tca gct atg ctg act ttt gag l Ser Cys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu 385 39ct ctc tca gaa acc gca gaa ttt gct aga aag tgg gtt ccc ttt tgt r Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys 44aag aag cac aat att gaa cca aga gct cca gaa ttt tac ttt gct caa s Lys His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln 423ta gat tac ctg aag gac aaa att caa cct tca ttt gtt aag gaa s Ile Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu 435 44ga cgc gca atg aag agg gag tat gaa gaa ttc aaa gta aga atc aat g Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn 456tt gtt gcc aaa gca cag aaa gtg cct gaa gag ggg tgg acc atg a Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met 465 47ct gat gga act gca tgg cct ggg aat aat cct agg gac cat cct ggc a Asp Gly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly 489tg att cag gtt ttc ttg ggg cac agt ggt ggg ctc gac act gat gga t Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly 55gag tta cca cgt ctt gtc tat gtc tct cgt gaa aag aga cca ggc n Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly 5525 ttt cag cat cac aag aag gct ggt gca atg aat gcg ctg att cgt gta e Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val 534ct gtg ctg aca aat ggt gcc tat ctt ctc aat gtg gat tgc gac r Ala Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp 545 55at tac
ttc aat agc agc aaa gct ctt aga gaa gca atg tgc ttc atg s Tyr Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met 567tg gat ccg gct cta gga agg aaa act tgt tat gta caa ttt cca cag t Asp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln 589tt gat ggc att gac ttg cac gat cga tat gct aat cgg aac ata 2 Phe Asp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile 595 6gtt ttc ttt gat atc aac atg aaa ggt ctg gat ggc att cag ggt cca 2 Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro 662ac gtg gga aca gga tgc tgt ttc aat aga cag gct ttg tat gga 2 Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly 625 63ac gat cct gtt ttg act gaa gct gat ctg gag cca aac att gtt att 2 Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Ile 645ag agc tgc tgt ggt aga agg aag aaa aag aac aag agt tat atg gat 2 Ser Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys Ser Tyr Met Asp 667aa agc cgt att atg aag aga aca gaa tct tca gct ccc atc ttc 2244 Ser Gln Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe 675 68at atg gaa gac atc gaa gag ggt att gaa ggt tac gag gat gaa agg 2292 Asn Met Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg 69gtg ctt atg tcc cag agg aaa ttg gag aaa cgc ttt ggt cag tct 234al Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser 77att ttc att gca tcc acc ttt atg aca caa ggt ggc ata cca cct 2388 Pro Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro 723ca aca aac cca gct tct cta cta aag gaa gct atc cat gtc atc agt 2436 Ser Thr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser 745ga tat gag gac aaa act gaa tgg gga aaa gag att ggc tgg atc 2484 Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile 755 76at ggt tca gta acg gag gat att ctg act ggg ttt aaa atg cat gca 2532 Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala 778gc tgg caa tca atc tac tgc atg cca cca cga cct tgt ttc aag 258ly Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys 785 79gt tct gca cca atc aat ctt tcc gat cgt ctt aat cag gtg ctc cgt 2628 Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg 88tgg gct ctt ggg tca gtg gaa att ctg ctt agt aga cat tgt cct atc 2676 Trp Ala Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile 823at ggt tac aat gga cga ttg aag ctt ttg gag agg ctg gct tac 2724 Trp Tyr Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr 835 84tc aac act att gta tat cca atc aca tcc att ccg ctt att gcc tat 2772 Ile Asn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Ile Ala Tyr 856tg ctt ccc gct atc tgc ctc ctt acc aat aaa ttt atc att cct 282al Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro 865 87ag att agc aat tat gct ggg atg ttc ttc att ctt ctt ttc gcc tcc 2868 Glu Ile Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser 889tt ttt gcc act ggt ata ttg gag ctt aga tgg agt ggt gtt ggc att 29Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile 99gat tgg tgg aga aat gag cag ttt tgg gtt att ggt ggc acc tct 2964 Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser 9925 gcc cat ctc ttc gca gtg ttc cag ggt ctg ctg aaa gtg ttg gct ggg 3 His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly 934at acc aac ttc aca gtt acc tca aag gca tct gat gag gat ggc 3 Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly 945 95ac ttt gct gag cta tat gtg ttc aag tgg acc agt ttg ctc att cct 3 Phe Ala Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro 967cg acc act gtt ctt gtc att aac ctg gtc gga atg gtg gca gga att 3 Thr Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile 989at gcc att aac agt ggc tac caa tcc tgg ggt ccg ctc ttt gga 32Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly 995 ctg ttc ttc tcg atc tgg gtg atc ctc cat ctc tac ccc ttc ctc 3252 Lys Leu Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu aag ggt ctc atg gga agg cag aac cgc aca cca aca atc gtc att gtc 33Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val 3tgg tcc atc ctt ctt gca tct atc ttc tcc ttg ctg tgg gtg aag atc 3348 Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile 45 55 gat cct ttc atc tcc ccg aca cag aaa gct gct gcc ttg ggg caa tgt 3396 Asp Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys 65 c gtc aac tgc tgatcgagac agtgactctt atttgaagag gctcaatcaa 3448 Gly Val Asn Cys tctgcccc ctcgtgtaaa tacctgagga ggctagatgg gaattccttt tgttgtaggt 35atggat ttgcatctaa gttatgcctc tgttcattag cttcttccgt gccggtgctg 3568 ctgcggacta agaatcacgg agcctttcta ccttccatgt agcgccagcc agcagcgtaa 3628 gatgtgaatt ttgaagtttt gttatgcgtg cagtttattg ttttagagta aattatcatt 3688 tgtttgtggg aactgttcac acgagcttat aatggcaatg ctgttattta aaaaaaaaaa 3748 aaaaa 3753 34 T Zea mays 34 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu Val Met Ile Arg His Asp Gly Asp Val Pro Gly Ser Ala Lys Pro 2 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Ser Val 35 4y Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9o Arg Val His Gly Asp Glu Asp Glu Glu Asp Val Asp Asp Leu Asp Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys Gly Pro Glu Trp Gln Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Glu Pro His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Met Gln Val 222sn Lys Tyr Pro Glu Ala Arg Gly Gly Asp Met Glu Gly Thr Gly 225 234sn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu 245 25er Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Val 267le Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg 275 28al Ser His Pro Val Arg Asp Ala Tyr Gly Leu Trp Leu Val Ser Val 29Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro 33Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu 325 33rg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val 345al Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala 355 36sn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp Lys Val 378ys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser 385 39Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys 44His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys 423sp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg 435 44rg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala 456al Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala 465 478ly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met 485 49le Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn 55Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe 5525 Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser 534al Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His 545 556he Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met 565 57sp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg 589sp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val 595 6Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val 662al Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr 625 634ro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Ile Lys 645 65er Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys Ser Tyr Met Asp Ser 667er Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn 675 68et Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser 69Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro 77Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser 725 73hr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys 745yr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr 755 76ly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg 778rp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly 785 79Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp 88Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp 823ly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile 835 84sn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Ile Ala Tyr Cys 856eu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu 865 878er Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile 885 89he Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu 99Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala 9925 His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile 934hr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp 945 956la Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro 965 97hr Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser 989la Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys 995 Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp 3r Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp 5Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly 65 l Asn Cys 25 DNA Zea mays 35 atggcggcca acaaggggat ggtgg 25 36 25 DNA Zea mays 36 tcagcagttg acgccacatt gcccc 25 37 3969 DNA Zea mays CDS ((34cttctccctc gtcggtgcgg cgtggcgcgg ctcggcgttc ggtgagaaac cactcggggg 6gatct gctgctagag tgagaggagc tacggtcagt atcctctgcc ttcgtcggcg gaagtgg aggggaggaa gcg atg gag gcg agc gcc ggg ctg gtg gcc ggc Glu Ala Ser Ala Gly Leu Val Ala Gly tcc cac aac cgc aac gag ctc gtc gtc atc cgc cgc gac ggc gat ccc 22is Asn Arg Asn Glu Leu Val Val Ile Arg Arg Asp Gly Asp Pro 5 ggg ccg aag ccg ccg cgg gag cag aac ggg cag gtg tgc cag att tgc 269 Gly Pro Lys Pro Pro Arg Glu Gln Asn Gly Gln Val Cys Gln Ile Cys 3 ggc gac gac gtc ggc ctt gcc ccc ggc ggg gac ccc ttc gtg gcg tgc 3Asp Asp Val Gly Leu Ala Pro Gly Gly Asp Pro Phe Val Ala Cys 45 5c gag tgc gcc ttc ccc gtc tgc cgg gac tgc tac gaa tac gag cgc 365 Asn Glu Cys Ala Phe Pro Val Cys Arg Asp Cys Tyr Glu Tyr Glu Arg 6 cgg gag ggc acg cag aac tgc ccc cag tgc aag act cga tac aag cgc 4Glu Gly Thr Gln Asn Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg 75 8 ctc aag ggc tgc caa cgt gtg acc ggt gac gag gag gag gac ggc gtc 46ys Gly Cys Gln Arg Val Thr Gly Asp Glu Glu Glu Asp Gly Val 95 gat gac ctg gac aac gag ttc aac tgg gac ggc cat gac tcg cag tct 5Asp Leu Asp Asn Glu Phe Asn Trp Asp Gly His Asp Ser Gln Ser gcc gag tcc atg ctc tac ggc cac atg agc tac ggc cgt gga ggt 557 Val Ala Glu Ser Met Leu Tyr Gly His Met Ser Tyr Gly Arg Gly Gly cct aat ggc gcg cca caa gct ttc cag ctc aac ccc aat gtt cca 6Pro Asn Gly Ala Pro Gln Ala Phe Gln Leu Asn Pro Asn Val Pro ctc acc aac ggg caa atg gtg gat gac atc cca ccg gag cag cac 653 Leu Leu Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His gcg ctg gtg cct tct ttc atg ggt ggt ggg gga aag agg ata cat ccc 7Leu Val Pro Ser Phe Met Gly Gly Gly Gly Lys Arg Ile His Pro cct tat gcg gat ccc agc tta cct gtg caa ccc agg tct atg gac 749 Leu Pro Tyr Ala Asp Pro Ser Leu Pro Val Gln Pro Arg Ser Met Asp 2tcc aag gat ctt gct gca tat ggg tat ggt agt gtt gct tgg aag 797 Pro Ser Lys Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp Lys 22cgg atg gag aat tgg aag cag aga caa gag agg atg cac cag acg 845 Glu Arg Met Glu Asn Trp Lys Gln Arg Gln Glu Arg Met His Gln Thr 223at gat ggt ggt ggt gat gat ggt gac gat gct gat cta cca cta 893 Gly Asn Asp Gly Gly Gly Asp Asp Gly Asp Asp Ala Asp Leu Pro Leu 235 245at gaa gca aga caa caa ctg tcc agg aaa att cca ctt
cca tca 94sp Glu Ala Arg Gln Gln Leu Ser Arg Lys Ile Pro Leu Pro Ser 255 26gc cag att aat cca tat agg atg att atc att att cgg ctt gtg gtt 989 Ser Gln Ile Asn Pro Tyr Arg Met Ile Ile Ile Ile Arg Leu Val Val 278gg ttc ttc ttc cac tac cga gtg atg cat ccg gtg aat gat gca u Gly Phe Phe Phe His Tyr Arg Val Met His Pro Val Asn Asp Ala 285 29tt gct ttg tgg ctc ata tct gtt atc tgt gaa atc tgg ttt gcc atg e Ala Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe Ala Met 33tgg att ctt gat caa ttc cca aag tgg ttc cct att gag aga gag r Trp Ile Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu 3325 33ac cta gac cgg ctg tca ctg agg ttc gac aag gaa ggc cag cca r Tyr Leu Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly Gln Pro 335 34ct caa ctt gct cca att gat ttc ttt gtc agt acg gtt gat ccc tta r Gln Leu Ala Pro Ile Asp Phe Phe Val Ser Thr Val Asp Pro Leu 356aa cct cct ttg gtc aca aca aat act gtt cta tct atc ctt tcg s Glu Pro Pro Leu Val Thr Thr Asn Thr Val Leu Ser Ile Leu Ser 365 37tg gat tat cct gtt gat aag gtt tct tgc tat gtt tct gat gat ggt l Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly 389ca atg cta acg ttt gaa gca tta tct gaa aca tct gaa ttt gca a Ala Met Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala 395 44aaa tgg gtt cct ttc tgc aaa cgg tac aat att gaa cct cgc gct s Lys Trp Val Pro Phe Cys Lys Arg Tyr Asn Ile Glu Pro Arg Ala 4425 cca gag tgg tac ttc caa cag aag ata gac tac ttg aaa gac aag gtg o Glu Trp Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val 434ca aac ttt gtt agg gag agg aga gca atg aag aga gag tat gag a Ala Asn Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu 445 45aa ttc aag gtg aga atc aat gcc tta gtt gcc aaa gcc cag aaa gtt u Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Val 467aa gaa gga tgg aca atg caa gat gga acc ccc tgg cct gga aac o Glu Glu Gly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn 475 489tt cgt gat cat cct gga atg att cag gtc ttc ctt ggc caa agc n Val Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly Gln Ser 495 5gga ggc ctt gac tgt gag gga aat gaa ctg cca cga ttg gtt tat gtt y Gly Leu Asp Cys Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val 552ga gag aaa cga cca ggc tat aac cat cat aag aaa gct ggt gct r Arg Glu Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala Gly Ala 525 53tg aat gca ttg gtc cga gtc tct gct gta cta aca aat gct cca tat t Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr 545ta aac ttg gat tgt gat cac tac atc aac aac agc aag gct ata u Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Ile 555 567aa gca atg tgt ttt atg atg gac cct tta cta gga aag aag gtt s Glu Ala Met Cys Phe Met Met Asp Pro Leu Leu Gly Lys Lys Val 575 58gc tat gta cag ttc cct caa aga ttt gat ggg att gat cgc cat gac s Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg His Asp 59tat gct aac cgg aat gtt gtc ttt ttt gat atc aac atg aaa ggt g Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met Lys Gly 66gat ggt att cag ggt cca att tat gtt ggt act gga tgt gta ttt 2 Asp Gly Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys Val Phe 623gg cag gca tta tat ggt tat gat gcc ccc aaa aca aag aag cca 2 Arg Gln Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro 635 645ca agg act tgc aac tgc tgg ccc aag tgg tgc ttt tgc tgt tgc 2 Ser Arg Thr Cys Asn Cys Trp Pro Lys Trp Cys Phe Cys Cys Cys 655 66gc ttt ggc aat agg aag caa aag aag act acc aaa ccc aaa aca gag 2 Phe Gly Asn Arg Lys Gln Lys Lys Thr Thr Lys Pro Lys Thr Glu 678aa aag tta tta ttt ttc aag aaa gaa gag aac caa tcc cct gca 2237 Lys Lys Lys Leu Leu Phe Phe Lys Lys Glu Glu Asn Gln Ser Pro Ala 685 69at gct ctt ggt gaa att gac gaa gct gct cca gga gct gag aat gaa 2285 Tyr Ala Leu Gly Glu Ile Asp Glu Ala Ala Pro Gly Ala Glu Asn Glu 77gcc ggt att gta aat caa caa aaa tta gaa aag aaa ttt ggc caa 2333 Lys Ala Gly Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln 7725 73ct gtt ttt gtt aca tcc aca ctt ctc gag aat ggt gga acc ttg 238er Val Phe Val Thr Ser Thr Leu Leu Glu Asn Gly Gly Thr Leu 735 74ag agt gca agt cct gct tct ctt ttg aaa gaa gct ata cat gtc att 2429 Lys Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile 756gt ggt tat gaa gac aag aca gac tgg gga aaa gag att ggc tgg 2477 Ser Cys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Lys Glu Ile Gly Trp 765 77tc tat gga tca gtt aca gaa gat att cta act ggt ttc aag atg cat 2525 Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His 789at ggt tgg cgg tca att tac tgc ata cct aaa cgg gtt gca ttc 2573 Cys His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Val Ala Phe 795 88ggt tct gca cct ctg aat ctt tca gat cgt ctt cac cag gtg ctt 262ly Ser Ala Pro Leu Asn Leu Ser Asp Arg Leu His Gln Val Leu 8825 cgg tgg gct ctt ggg tct att gag atc ttc ttc agc aat cat tgc cct 2669 Arg Trp Ala Leu Gly Ser Ile Glu Ile Phe Phe Ser Asn His Cys Pro 834gg tat ggg tat ggt ggc ggt ctg aaa ttt ttg gaa aga ttt tcc 27Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser 845 85ac atc aac tcc atc gtg tat cct tgg aca tct att ccc ctc ttg gct 2765 Tyr Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala 867gt aca ttg cct gcc atc tgt tta ttg aca ggg aaa ttt atc act 28Cys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr 875 889ag ctg aat aat gtt gcc agc ctg tgg ttc atg tca ctt ttt atc 286lu Leu Asn Asn Val Ala Ser Leu Trp Phe Met Ser Leu Phe Ile 895 9tgc att ttt gct acg agc atc cta gaa atg aga tgg agt ggt gtt gga 29Ile Phe Ala Thr Ser Ile Leu Glu Met Arg Trp Ser Gly Val Gly 992at gac tgg tgg agg aat gag cag ttc tgg gtc att gga ggt gtg 2957 Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val 925 93cc tca cac ctc ttt gct gtg ttc cag gga ctt ctc aag gtc ata gct 3 Ser His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Ile Ala 945tt gat aca agc ttc acc gtg aca tca aag ggt gga gat gat gag 3 Val Asp Thr Ser Phe Thr Val Thr Ser Lys Gly Gly Asp Asp Glu 955 967tc tca gag cta tat aca ttc aaa tgg act acc tta ttg ata cct 3 Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro 975 98ct acc acc ttg ctt cta ttg aac ttc att ggt gtg gtc gct ggc gtt 3 Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala Gly Val 99 aat gcg atc aat aac gga tat gag tca tgg ggc ccc ctc ttt ggg 3 Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly aag cta ttc ttt gca ttt tgg gtg att gtc cat ctt tat ccc ttt ctc 3245 Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu 25 a ggt ttg gtt gga agg caa aac agg aca cca acg att gtc atc gtc 3293 Lys Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val 4g tcc att ctg ctg gct tca atc ttc tcg ctc ctt tgg gtt cgg att 334er Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile 6gat cct ttc ctt gcg aag gat gat ggt ccg ctt ctt gag gag tgt ggt 3389 Asp Pro Phe Leu Ala Lys Asp Asp Gly Pro Leu Leu Glu Glu Cys Gly 75 g gat tgc aac taggatgtca gtgcatcagc tcccccaatc tgcatatgct 344sp Cys Asn aagtatat tttctggtgt ttgtccccat attcagtgtc tgtagataag agacatgaaa 35ccaagt ttcttttgat ccatggtgaa cctacttaat atctgagaga tatactgggg 356tggag gctgcggcaa tccttgtgca gttgggccgt ggaatacagc atatgcaagt 362attgt gcagcattct ttattacttg gtcgcaatat agatgggctg agccgaacag 368tattt tgattctgca ctgctcccgt gtacaaactt ggttctcaat aaggcaggca 374gcatc tgccagtgga acagagcaac ctgcacatta tttatgtatg cctgttcatt 38ggcttg ttcattacat gttcgtctat actagaaaaa acagaatatt agcattaatc 386ttaat taaagtatgt aaatgcgcct gttttttgtt gtgtactgta atcatctgag 392tttgt gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3969 38 T Zea mays 38 Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu Val Val Ile Arg Arg Asp Gly Asp Pro Gly Pro Lys Pro Pro Arg 2 Glu Gln Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Asp Val Gly Leu 35 4a Pro Gly Gly Asp Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro 5 Val Cys Arg Asp Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr Gln Asn 65 7 Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Leu Lys Gly Cys Gln Arg 85 9l Thr Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Asp Asn Glu Asn Trp Asp Gly His Asp Ser Gln Ser Val Ala Glu Ser Met Leu Gly His Met Ser Tyr Gly Arg Gly Gly Asp Pro Asn Gly Ala Pro Ala Phe Gln Leu Asn Pro Asn Val Pro Leu Leu Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His Ala Leu Val Pro Ser Phe Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Tyr Ala Asp Pro Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys Asp Leu Ala 2Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met Glu Asn Trp 222ln Arg Gln Glu Arg Met His Gln Thr Gly Asn Asp Gly Gly Gly 225 234sp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu Ala Arg Gln 245 25ln Leu Ser Arg Lys Ile Pro Leu Pro Ser Ser Gln Ile Asn Pro Tyr 267et Ile Ile Ile Ile Arg Leu Val Val Leu Gly Phe Phe Phe His 275 28yr Arg Val Met His Pro Val Asn Asp Ala Phe Ala Leu Trp Leu Ile 29Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile Leu Asp Gln 33Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg Leu 325 33er Leu Arg Phe Asp Lys Glu Gly Gln Pro Ser Gln Leu Ala Pro Ile 345he Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Val 355 36hr Thr Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp 378al Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe 385 39Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp Val Pro Phe 44Lys Arg Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Gln 423ys Ile Asp Tyr Leu Lys Asp Lys Val Ala Ala Asn Phe Val Arg 435 44lu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile 456la Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr 465 478ln Asp Gly Thr Pro Trp Pro Gly Asn Asn Val Arg Asp His Pro 485 49ly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly Leu Asp Cys Glu 55Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro 5525 Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg 534er Ala Val Leu Thr Asn Ala Pro Tyr Leu Leu Asn Leu Asp Cys 545 556is Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala Met Cys Phe 565 57et Met Asp Pro Leu Leu Gly Lys Lys Val Cys Tyr Val Gln Phe Pro 589rg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ala Asn Arg Asn 595 6Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly 662le Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr 625 634yr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg Thr Cys Asn 645 65ys Trp Pro Lys Trp Cys Phe Cys Cys Cys Cys Phe Gly Asn Arg Lys 667ys Lys Thr Thr Lys Pro Lys Thr Glu Lys Lys Lys Leu Leu Phe 675 68he Lys Lys Glu Glu Asn Gln Ser Pro Ala Tyr Ala Leu Gly Glu Ile 69Glu Ala Ala Pro Gly Ala Glu Asn Glu Lys Ala Gly Ile Val Asn 77Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser Ser Val Phe Val Thr 725 73er Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys Ser Ala Ser Pro Ala 745eu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp 755 76ys Thr Asp Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr 778sp Ile Leu Thr Gly Phe Lys Met His Cys His Gly Trp Arg Ser 785 79Tyr Cys Ile Pro Lys Arg Val Ala Phe Lys Gly Ser Ala Pro Leu 88Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly Ser 823lu Ile Phe Phe Ser Asn His Cys Pro Leu Trp Tyr Gly Tyr Gly 835 84ly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr Ile Asn Ser Ile Val 856ro Trp Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Leu Pro Ala 865 878ys Leu Leu Thr Gly Lys Phe Ile Thr Pro Glu Leu Asn Asn Val 885 89la Ser Leu Trp Phe Met Ser Leu Phe Ile Cys Ile Phe Ala Thr Ser 99Leu Glu Met Arg Trp Ser Gly Val Gly Ile Asp Asp Trp Trp Arg 9925 Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ser His Leu Phe Ala 934he Gln Gly Leu Leu Lys Val Ile Ala Gly Val Asp Thr Ser Phe 945 956al Thr Ser Lys Gly Gly Asp Asp Glu Glu Phe Ser Glu Leu Tyr 965 97hr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu Leu 989sn Phe Ile Gly Val Val Ala Gly Val Ser Asn Ala Ile Asn Asn 995 Tyr Glu
Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Val Gly Arg 3n Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser Ile Leu Leu Ala 5Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Leu Ala Lys 65 p Asp Gly Pro Leu Leu Glu Glu Cys Gly Leu Asp Cys Asn 839 25 DNA Zea mays 39 atggaggcga gcgccgggct ggtgg 25 4A Zea mays 4tgcaa tccaaaccac actcc 25 4DNA Zea mays CDS ((34gcagcagcag caccaccact gcgcggcatt gcagcgagca agcgggaggg atctggggca 6gcggt cgctgccgct gccgctcgga tctagagggc cgcacgggct gattgccctc cggcctc gtcggtgtcg gtggagtgtg aatcggtgtg tgtaggagga gcgcggag gcg gcc aac aag ggg atg gtg gca ggc tct cac aac cgc aac gag 226 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu gtc atg atc cgc cac gac ggc gac gcg cct gtc ccg gct aag ccc 274 Phe Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 2 acg aag agt gcg aat ggg cag gtc tgc cag att tgt ggc gac act gtt 322 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 4c gtt tca gcc act ggt gat gtc ttt gtt gcc tgc aat gag tgt gcc 37al Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 ttc cct gtc tgc cgc cct tgc tat gag tac gag cgc aag gaa ggg aac 4Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 caa tgc tgc cct cag tgc aag act aga tac aag aga cag aaa ggt agc 466 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9t cga gtt cat ggt gat gat gag gag gaa gat gtt gat gac ctg gac 5Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp gaa ttc aac tat aag caa ggc aat ggg aag ggc cca gag tgg cag 562 Asn Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln caa gga gat gac gct gat ctg tct tca tct gct cgc cat gac cca 6Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro cat cgg att cca cgc ctt aca agt gga caa cag ata tct gga gag 658 His His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu atc cct gat gca tcc cct gac cgt cat tct atc cgc agt cca aca tcg 7Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser tat gtt gat cca agc gtt cca gtt cct gtg agg att gtg gac ccc 754 Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro aag gac ttg aat tcc tat ggg ctt aat agt gtt gac tgg aag gaa 8Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2gtt gag agc tgg agg gtt aaa cag gac aaa aat atg ttg caa gtg 85al Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 222at aaa tat cca gag gct aga gga gac atg gag ggg act ggc tca 898 Thr Asn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser 225 234ga gaa gat atg caa atg gtt gat gat gca cgc cta cct ttg agc 946 Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 25gc att gtg cca att tcc tca aac cag ctc aac ctt tac cgg ata gta 994 Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 267tt ctc cgt ctt atc atc ctg tgc ttc ttc ttc caa tat cgt atc e Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 28gt cat cca gtg cgt aat gct tat gga ttg tgg cta gta tct gtt atc r His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 29gag gtc tgg ttt gcc ttg tcc tgg ctt cta gat cag ttc cca aaa s Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys 33tgg tat cca atc aac cgt gag aca tat ctc gac agg ctt gca ttg agg p Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 33at gat aga gag gga gag cca tca cag ctg gct ccc att gat gtc ttt r Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 345gt aca gtg gat cca ttg aag gaa cct cca ctg atc aca gcc aac l Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 36ct gtt ttg tcc att ctt gct gtg gat tac cct gtt gac aaa gtg tca r Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 378at gtt tct gat gat ggc tca gct atg ctg act ttt gag tct ctc s Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu 385 39gaa act gcc gaa ttt gct aga aag tgg gtt ccc ttt tgt aag aag r Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 44aat att gaa cca aga gct cca gaa ttt tac ttt gct caa aaa ata s Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 423ac ctg aag gac aaa att caa cct tca ttt gtt aag gaa aga cga p Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 44ca atg aag aga gag tat gaa gaa ttc aaa ata aga atc aat gcc ctt a Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 456cc aaa gca cag aaa gtg cct gaa gag ggg tgg acc atg gct gat l Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp 465 478ct gct tgg cct ggg aat aac cct agg gac cat cct ggc atg att y Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 49ag gtg ttc ttg ggg cac agt ggt ggg ctt gac act gat gga aat gaa n Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 55cca cgt ctt gtc tat gtc tct cgt gaa aag aga cca ggc ttt cag u Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln 5525 cat cac aag aag gct ggt gca atg aat gca ctg att cgt gta tct gct s His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 534tg aca aat ggt gcc tat ctt ctc aat gtg gat tgt gac cat tac l Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr 545 556at agc agc aaa gct ctt aga gaa gca atg tgc ttc atg atg gat e Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 57ca gct cta gga agg aaa act tgt tat gta caa ttt cca caa aga ttt o Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 589gc att gac ttg cac gat cga tat gct aat agg aac ata gtc ttc 2 Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 6ttt gat atc aac atg aaa ggt cta gat ggc att cag ggt cca gtc tat 2 Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 662ga aca gga tgc tgt ttc aat agg cag gct ttg tat gga tat gat 2 Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp 625 634tt ttg act gaa gct gat ctg gaa cct aac att gtt gtt aag agc 2 Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 65gc tgt ggt aga agg aag aga aag aac aag agt tat atg gat agt caa 2 Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 667gt att atg aag aga aca gaa tct tca gct ccc atc ttt aac atg 2242 Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 68aa gac atc gag gag ggt att gaa ggt tat gag gat gaa agg tca gtg 229sp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 69atg tcc cag agg aaa ttg gag aaa cgc ttt ggt cag tct cca atc 2338 Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile 77ttc att gca tcc acc ttt atg act caa ggt ggc ata cca cct tca aca 2386 Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 73ac cca gct tct cta ctg aag gaa gct atc cat gtt atc agc tgt ggg 2434 Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 745ag gac aaa act gaa tgg gga aaa gag att ggc tgg atc tat ggt 2482 Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 76ca gtt aca gag gat att ctg act ggg ttt aaa atg cat gca aga ggc 253al Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 778aa tca atc tac tgc atg cca cca cga cct tgt ttc aag ggt tct 2578 Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser 785 79cca atc aat ctt tct gat cgt ctt aat cag gtg ctc cgt tgg gct 2626 Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 88ggg tca gtg gaa att ctg ctt agc aga cat tgt cct ata tgg tat 2674 Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 823ac aat ggg cga ttg aag ctt ttg gag agg ctg gct tac att aac 2722 Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 84cc att gtt tat cca atc aca tct gtt ccg ctt atc gcc tat tgt gtg 277le Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 856ct gct atc tgt ctt ctt acc aat aaa ttt atc att cct gag att 28Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile 865 878at tat gct gga atg ttc ttc att ctt ctt ttt gcc tcc att ttc 2866 Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 89ca act ggt ata ttg gag ctc aga tgg agt ggt gtt ggc att gaa gat 29Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 99tgg aga aat gag cag ttt tgg gtt att ggt ggc acc tct gcc cat 2962 Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 9925 ctc ttc gcg gtg ttc cag ggt ctg ctg aaa gtg ttg gct ggg att gat 3 Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 934ac ttc aca gtt acc tca aag gca tct gat gag gat ggc gac ttt 3 Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 945 956ag cta tat gtg ttc aag tgg acc agt ttg ctc atc cct ccg acc 3 Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 97ct gtt ctt gtc att aac ctg gtc gga atg gtg gca gga att tcg tat 3 Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 989tt aac agc ggc tac caa tcc tgg ggt ccg ctc ttt gga aag ctg 32Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 ttc tcg atc tgg gtg atc ctc cat ctc tac ccc ttc ctc aag ggt 325he Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly ctc atg ggc agg cag aac cgc acg cca aca atc gtc atc gtt tgg tcc 3298 Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser 3c ctc ctt gcg tct atc ttc tcc ttg ctg tgg gtg aag atc gat cct 3346 Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 5ttc atc tcc ccg aca cag aaa gct gcc gcc ttg ggg caa tgt ggt gtg 3394 Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 65 c tgc tgatccagat tgtgactctt atctgaagag gctcagccaa agatctgccc 345ys cctcgtgtaa atacctgagg gggctagatg ggaatttttt gttgtagatg aggatggatc 35tccaag ttatgcctct gtttattagc ttcttcggtg ccggtgctgc tgcagacaat 357agcct ttctaccttg cttgtagtgc tggccagcag cgtaaattgt gaattctgca 363ttata cgtggtgttt attgttttag agtaaattat catttgtttg aggtaactat 369cgaac tatatggcaa tgctgttatt taaaa 3725 42 T Zea mays 42 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 2 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 4y Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9o Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 222sn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser 225 234ly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 25rg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 267le Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 28er His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 29Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys 33Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 33yr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 345er Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 36hr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 378yr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu 385 39Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 44Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 423yr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 44la Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 456la Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp 465 478hr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 49ln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 55Pro Arg Leu Val Tyr Val Ser Arg Glu Lys
Arg Pro Gly Phe Gln 5525 His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 534eu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr 545 556sn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 57ro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 589ly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 6Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 662ly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp 625 634al Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 65ys Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 667rg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 68lu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 69Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile 77Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 73sn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 745lu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 76er Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 778ln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser 785 79Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 88Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 823yr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 84hr Ile Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 856ro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile 865 878sn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 89la Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 99Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 9925 Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 934sn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 945 956lu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 97hr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 989le Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser 3e Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 5Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 65 n Cys 43 25 DNA Zea mays 43 atggcggcca acaaggggat ggtgg 25 44 25 DNA Zea mays 44 tcagcagttc acaccacatt gcccc 25 45 38Zea mays CDS (23496) 45 ccacagctca tataccaaga gccggagcag cttagcgcag cccagagcgg cgccgcgcca 6aaccc ccacccgcca cagccgcgtg cgcatgtgag cggtcgccgc ggccgggaga gaggagg ggaggactac gtgcatttcg ctgtgccgcc gccgcggggt tcgtgcgcga agatccg gcggggcggg gcggggggcc tgag atg gag gct agc gcg ggg ctg 235 Met Glu Ala Ser Ala Gly Leu gcc ggc tcg cat aac cgg aac gag ctg gtg gtg atc cgc cgc gac 283 Val Ala Gly Ser His Asn Arg Asn Glu Leu Val Val Ile Arg Arg Asp ag tcg gga gcc gcg ggc ggc ggc gcg gcg cgc cgg gcg gag gcg 33lu Ser Gly Ala Ala Gly Gly Gly Ala Ala Arg Arg Ala Glu Ala 25 3g tgc cag ata tgc ggc gac gag gtc ggg gtg ggc ttc gac ggg gag 379 Pro Cys Gln Ile Cys Gly Asp Glu Val Gly Val Gly Phe Asp Gly Glu 4 55 ccc ttc gtg gcg tgc aac gag tgc gcc ttc ccc gtc tgc cgc gcc tgc 427 Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro Val Cys Arg Ala Cys 6 tac gag tac gag cgc cgc gag ggc tcg caa gcg tgc ccg cag tgc agg 475 Tyr Glu Tyr Glu Arg Arg Glu Gly Ser Gln Ala Cys Pro Gln Cys Arg 75 8c cgc tac aag cgc ctc aag ggc tgc ccg cgg gtg gcc ggc gac gag 523 Thr Arg Tyr Lys Arg Leu Lys Gly Cys Pro Arg Val Ala Gly Asp Glu 9ag gac ggc gtc gac gac ctg gag ggc gag ttc ggc ctg cag gac 57lu Asp Gly Val Asp Asp Leu Glu Gly Glu Phe Gly Leu Gln Asp gcc gcc cac gag gac gac ccg cag tac gtc gcc gag tcc atg ctc 6Ala Ala His Glu Asp Asp Pro Gln Tyr Val Ala Glu Ser Met Leu agg gcg cag atg agc tac ggc cgc ggc ggc gac gcg cac ccc ggc ttc 667 Arg Ala Gln Met Ser Tyr Gly Arg Gly Gly Asp Ala His Pro Gly Phe ccc gtc ccc aac gtg ccg ctc ctc acc aac ggc cag atg gtt gat 7Pro Val Pro Asn Val Pro Leu Leu Thr Asn Gly Gln Met Val Asp atc ccg ccg gag cag cac gcg ctc gtg ccg tcc tac atg agc ggc 763 Asp Ile Pro Pro Glu Gln His Ala Leu Val Pro Ser Tyr Met Ser Gly ggc ggc ggg ggc aag agg atc cac ccg ctc cct ttc gca gat ccc 8Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Phe Ala Asp Pro ctt cca gtg caa ccg aga tcc atg gac ccg tcc aag gat ctg gcc 859 Asn Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys Asp Leu Ala 22gcc tac gga tat ggc agc gtg gcc tgg aag gag aga atg gag ggc tgg 9Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met Glu Gly Trp 223ag aag cag gag cgc ctg cag cat gtc agg agc gag ggt ggc ggt 955 Lys Gln Lys Gln Glu Arg Leu Gln His Val Arg Ser Glu Gly Gly Gly 235 24at tgg gat ggc gac gat gca gat ctg cca cta atg gat gaa gct agg p Trp Asp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu Ala Arg 256ca ttg tcc aga aaa gtc cct ata tca tca agc cga att aat ccc n Pro Leu Ser Arg Lys Val Pro Ile Ser Ser Ser Arg Ile Asn Pro 265 27ac agg atg att atc gtt atc cgg ttg gtg gtt ttg ggt ttc ttc ttc r Arg Met Ile Ile Val Ile Arg Leu Val Val Leu Gly Phe Phe Phe 289ac tac cga gtg atg cat ccg gcg aaa gat gca ttt gca ttg tgg ctc s Tyr Arg Val Met His Pro Ala Lys Asp Ala Phe Ala Leu Trp Leu 33tct gta atc tgt gaa atc tgg ttt gcg atg tcc tgg att ctt gat e Ser Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile Leu Asp 3325 cag ttc cca aag tgg ctt cca atc gag aga gag act tac ctg gac cgt n Phe Pro Lys Trp Leu Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg 334ca cta agg ttt gac aag gaa ggt caa ccc tct cag ctt gct cca u Ser Leu Arg Phe Asp Lys Glu Gly Gln Pro Ser Gln Leu Ala Pro 345 35tc gac ttc ttt gtc agt acg gtt gat ccc aca aag gaa cct ccc ttg e Asp Phe Phe Val Ser Thr Val Asp Pro Thr Lys Glu Pro Pro Leu 367tc aca gcg aac act gtc ctt tcc atc ctt tct gtg gat tat ccg gtt l Thr Ala Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val 389ag gtc tcc tgc tat gtt tct gat gat ggt gct gca atg ctt acg u Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr 395 4ttt gaa gca ttg tct gaa aca tct gaa ttt gca aag aaa tgg gtt cct e Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp Val Pro 442gc aaa aag ttt aat atc gag cct cgt gct cct gag tgg tac ttc e Ser Lys Lys Phe Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe 425 43aa cag aag ata gac tac ctg aaa gac aag gtt gct gct tca ttt gtt n Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Ala Ala Ser Phe Val 445gg gag agg agg gcg atg aag aga gaa tac gag gaa ttc aag gta agg g Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg 467at gcc ttg gtt gca aaa gcc caa aag gtt cct gag gaa gga tgg e Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp 475 48ca atg caa gat gga agc ccc tgg cct gga aac aac gta cgc gat cat r Met Gln Asp Gly Ser Pro Trp Pro Gly Asn Asn Val Arg Asp His 49gga atg att cag gta ttc ctt ggc caa agt ggc ggt cgt gat gtg o Gly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly Arg Asp Val 55gga aat gag ttg cct cgc ctg gtt tat gtc tcg aga gaa aag agg u Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 523ca ggt tat aac cat cac aag aag gct ggt gcc atg aat gca ctg gtc o Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val 545tc tct gct gtc tta tca aat gct gca tac cta ttg aac ttg gac g Val Ser Ala Val Leu Ser Asn Ala Ala Tyr Leu Leu Asn Leu Asp 555 56gt gat cac tac atc aac aat agc aag gcc ata aaa gag gct atg tgt s Asp His Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala Met Cys 578tg atg gat cct ttg gtg ggg aag aaa gtg tgc tat gta cag ttc 2 Met Met Asp Pro Leu Val Gly Lys Lys Val Cys Tyr Val Gln Phe 585 59ct cag agg ttt gat ggt att gac aaa aat gat cga tac gct aac agg 2 Gln Arg Phe Asp Gly Ile Asp Lys Asn Asp Arg Tyr Ala Asn Arg 66aac gtt gtc ttt ttt gac atc aac atg aaa ggt ttg gac ggt att caa 2 Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln 623cc att tat gtg ggt act gga tgt gtt ttc aga cgg cag gca ctg 2 Pro Ile Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln Ala Leu 635 64at ggt tat gat gct cct aaa acg aag aag cca cca tca aga act tgc 22Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg Thr Cys 656gc tgg ccc aag tgg tgc ctc tct tgc tgc tgc agc agg aac aag 225ys Trp Pro Lys Trp Cys Leu Ser Cys Cys Cys Ser Arg Asn Lys 665 67at aaa aag aag act aca aaa cca aag acg gag aag aag aaa aga tta 2299 Asn Lys Lys Lys Thr Thr Lys Pro Lys Thr Glu Lys Lys Lys Arg Leu 689tt ttc aag aaa gca gaa aac cca tct cct gca tat gct ttg ggt gaa 2347 Phe Phe Lys Lys Ala Glu Asn Pro Ser Pro Ala Tyr Ala Leu Gly Glu 77gat gaa ggt gct cca ggt gct gat atc gag aag gcc gga atc gta 2395 Ile Asp Glu Gly Ala Pro Gly Ala Asp Ile Glu Lys Ala Gly Ile Val 7725 aat caa cag aaa cta gag aag aaa ttt ggg cag tct tct gtt ttt gtc 2443 Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser Ser Val Phe Val 734ca aca ctt ctt gag aac gga ggg acc ctg aag agc gca agt cca 249er Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys Ser Ala Ser Pro 745 75ct tct ctt ctg aag gaa gct ata cat gtt atc agc tgc ggc tac gaa 2539 Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu 767ac aag acc gac tgg gga aaa gag att ggc tgg att tac gga tcg atc 2587 Asp Lys Thr Asp Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Ile 789ag gat atc ttg act gga ttt aag atg cac tgc cat ggc tgg cgg 2635 Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys His Gly Trp Arg 795 8tct att tac tgc atc ccg aag cgg cct gca ttc aaa ggt tct gcg cct 2683 Ser Ile Tyr Cys Ile Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro 882ac ctt tcc gac cgt ctt cac cag gtc ctt cgc tgg gcc ctt ggg 273sn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly 825 83cc gtc gaa att ttc ttc agc aag cac tgc cca ctt tgg tac gga tac 2779 Ser Val Glu Ile Phe Phe Ser Lys His Cys Pro Leu Trp Tyr Gly Tyr 845gc ggc ggg cta aaa ttc ctg gaa agg ttt tct tat atc aac tcc atc 2827 Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr Ile Asn Ser Ile 867at ccc tgg acg tcc att cct ctc ctg gct tac tgt acc ttg cct 2875 Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Leu Pro 875 88cc atc tgc ctg ctc acg ggg aag ttt atc aca cca gag ctt acc aat 2923 Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr Pro Glu Leu Thr Asn 89gcc agt atc tgg ttc atg gca ctt ttc atc tgc atc tcc gtg acc 297la Ser Ile Trp Phe Met Ala Leu Phe Ile Cys Ile Ser Val Thr 99atc ctg gaa atg agg tgg agt ggc gtg gcc atc gac gac tgg tgg 3 Ile Leu Glu Met Arg Trp Ser Gly Val Ala Ile Asp Asp Trp Trp 923gg aac gag cag ttc tgg gtc atc gga ggc gtt tcg gcg cat ctg ttc 3 Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe 945tg ttc cag ggc ctg ctg aag gtg ttc gcc ggc atc gac acg agc 3 Val Phe Gln Gly Leu Leu Lys Val Phe Ala Gly Ile Asp Thr Ser 955 96tc acc gtg acg tcg aag gcc ggg gac gac gag gag ttc tcg gag ctg 3 Thr Val Thr Ser Lys Ala Gly Asp Asp Glu Glu Phe Ser Glu Leu 978cg ttc aag tgg acc acc ctg ctg ata ccc ccg acc acg ctc ctc 32Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu 985 99tg ctg aac ttc atc ggg gtg gtg gcc ggg atc tcg aac gcg atc aac 3259 Leu Leu Asn Phe Ile Gly Val Val Ala Gly Ile Ser Asn Ala Ile Asn c ggg tac gag tcg tgg ggc ccc ctg ttc ggg aag ctc ttc ttc gcc 33Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala 25 c tgg gtg atc gtc cac ctg tac ccg ttc ctc aag ggt ctg gtg ggg 3355 Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Val Gly 4agg cag aac agg acg ccg acg atc gtc atc gtc tgg tcc atc ctg ctg 34Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser Ile Leu Leu 55 c tcg atc ttc tcg ctc ctg tgg gtc cgc gtc gac ccg ttc ctc gcc 345er Ile Phe Ser Leu Leu Trp Val Arg Val Asp Pro Phe Leu Ala 7aag agc aac ggc ccg ctc ctg gag gag tgt ggc ctg gac tgc aac 3496 Lys Ser Asn Gly Pro Leu Leu Glu Glu Cys Gly Leu Asp Cys Asn 85 aagtgggg gccccctgtc actcgaagtt ctgtcacggg cgaattacgc ctgatttttt 3556 gttgttgttg ttgttggaat tctttgctgt agatagaaac cacatgtcca cggcatctct 36tgtcca ttggagcagg agagaggtgc ctgctgctgt ttgttgagta aattaaaagt 3676 tttaaagtta tacagtgatg cacattccag tgcccagtgt attccctttt tacagtctgt 3736 atattagcga caaaggacat attggttagg agtttgattc ttttgtaaaa aaaaaaaaaa 3796 aaaaaaaaaa aaaaaaa
38 Zea mays 46 Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu Val Val Ile Arg Arg Asp Arg Glu Ser Gly Ala Ala Gly Gly Gly 2 Ala Ala Arg Arg Ala Glu Ala Pro Cys Gln Ile Cys Gly Asp Glu Val 35 4y Val Gly Phe Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Ala Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Ser 65 7 Gln Ala Cys Pro Gln Cys Arg Thr Arg Tyr Lys Arg Leu Lys Gly Cys 85 9o Arg Val Ala Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Glu Glu Phe Gly Leu Gln Asp Gly Ala Ala His Glu Asp Asp Pro Gln Val Ala Glu Ser Met Leu Arg Ala Gln Met Ser Tyr Gly Arg Gly Asp Ala His Pro Gly Phe Ser Pro Val Pro Asn Val Pro Leu Leu Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His Ala Leu Pro Ser Tyr Met Ser Gly Gly Gly Gly Gly Gly Lys Arg Ile His Leu Pro Phe Ala Asp Pro Asn Leu Pro Val Gln Pro Arg Ser Met 2Pro Ser Lys Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp 222lu Arg Met Glu Gly Trp Lys Gln Lys Gln Glu Arg Leu Gln His 225 234rg Ser Glu Gly Gly Gly Asp Trp Asp Gly Asp Asp Ala Asp Leu 245 25ro Leu Met Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys Val Pro Ile 267er Ser Arg Ile Asn Pro Tyr Arg Met Ile Ile Val Ile Arg Leu 275 28al Val Leu Gly Phe Phe Phe His Tyr Arg Val Met His Pro Ala Lys 29Ala Phe Ala Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe 33Ala Met Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Leu Pro Ile Glu 325 33rg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly 345ro Ser Gln Leu Ala Pro Ile Asp Phe Phe Val Ser Thr Val Asp 355 36ro Thr Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile 378er Val Asp Tyr Pro Val Glu Lys Val Ser Cys Tyr Val Ser Asp 385 39Gly Ala Ala Met Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu 44Ala Lys Lys Trp Val Pro Phe Ser Lys Lys Phe Asn Ile Glu Pro 423la Pro Glu Trp Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp 435 44ys Val Ala Ala Ser Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu 456lu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln 465 478al Pro Glu Glu Gly Trp Thr Met Gln Asp Gly Ser Pro Trp Pro 485 49ly Asn Asn Val Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly 55Ser Gly Gly Arg Asp Val Glu Gly Asn Glu Leu Pro Arg Leu Val 5525 Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala 534la Met Asn Ala Leu Val Arg Val Ser Ala Val Leu Ser Asn Ala 545 556yr Leu Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys 565 57la Ile Lys Glu Ala Met Cys Phe Met Met Asp Pro Leu Val Gly Lys 589al Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Lys 595 6Asn Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met 662ly Leu Asp Gly Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys 625 634he Arg Arg Gln Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys 645 65ys Pro Pro Ser Arg Thr Cys Asn Cys Trp Pro Lys Trp Cys Leu Ser 667ys Cys Ser Arg Asn Lys Asn Lys Lys Lys Thr Thr Lys Pro Lys 675 68hr Glu Lys Lys Lys Arg Leu Phe Phe Lys Lys Ala Glu Asn Pro Ser 69Ala Tyr Ala Leu Gly Glu Ile Asp Glu Gly Ala Pro Gly Ala Asp 77Ile Glu Lys Ala Gly Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe 725 73ly Gln Ser Ser Val Phe Val Ala Ser Thr Leu Leu Glu Asn Gly Gly 745eu Lys Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His 755 76al Ile Ser Cys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Lys Glu Ile 778rp Ile Tyr Gly Ser Ile Thr Glu Asp Ile Leu Thr Gly Phe Lys 785 79His Cys His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Pro 88Phe Lys Gly Ser Ala Pro Leu Asn Leu Ser Asp Arg Leu His Gln 823eu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Phe Ser Lys His 835 84ys Pro Leu Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg 856er Tyr Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu 865 878la Tyr Cys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe 885 89le Thr Pro Glu Leu Thr Asn Val Ala Ser Ile Trp Phe Met Ala Leu 99Ile Cys Ile Ser Val Thr Gly Ile Leu Glu Met Arg Trp Ser Gly 9925 Val Ala Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly 934al Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val 945 956la Gly Ile Asp Thr Ser Phe Thr Val Thr Ser Lys Ala Gly Asp 965 97sp Glu Glu Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu 989ro Pro Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala 995 Ile Ser Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro 3e Leu Lys Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val 5Ile Val Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val 65 g Val Asp Pro Phe Leu Ala Lys Ser Asn Gly Pro Leu Leu Glu Glu 8Cys Gly Leu Asp Cys Asn 25 DNA Zea mays 47 atggaggcta gcgcggggct ggtgg 25 48 25 DNA Zea mays 48 tcagttgcag tccaggccac actcc 25 49 3746 DNA Zea mays CDS (32449) 49 ctaggatcaa aaccgtctcg ccgctgcaat aatcttttgt caattcttaa tccctcgcgt 6gcgac agcggaacca actcacgttg ccgcggcttc ctccatcggt gcggtgccct cttttct ctcgtccctc ctccccccgt atagttaagc cccgccccgc tactactact agcagca gcagcgctct cgcagcggga gatgcggtgt tgatccgtgc cccgctcgga 24ggact ggtgccggct ctgcccaggc cccaggctcc aggccagctc cctcgacgtt 3ggcgag ctcgcttgcc atg gag ggc gac gcg gac ggc gtg aag tcg ggg 353 Met Glu Gly Asp Ala Asp Gly Val Lys Ser Gly agg cgc ggt ggc gga cag gtg tgc cag atc tgc ggc gac ggc gtg ggc 4Arg Gly Gly Gly Gln Val Cys Gln Ile Cys Gly Asp Gly Val Gly 5 acc acg gcg gag ggg gac gtc ttc gcc gcc tgc gac gtc tgc ggg ttt 449 Thr Thr Ala Glu Gly Asp Val Phe Ala Ala Cys Asp Val Cys Gly Phe 3 ccg gtg tgc cgc ccc tgc tac gag tac gag cgc aag gac ggc acg cag 497 Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln 45 5g tgc ccc cag tgc aag acc aag tac aag cgc cac aag ggg agc ccg 545 Ala Cys Pro Gln Cys Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro 6 75 gcg atc cgt ggg gag gaa gga gac gac act gat gcc gat agc gac ttc 593 Ala Ile Arg Gly Glu Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe 8 aat tac ctt gca tct ggc aat gag gac cag aag cag aag att gcc gac 64yr Leu Ala Ser Gly Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp 95 aga atg cgc agc tgg cgc atg aac gtt ggg ggc agc ggg gat gtt ggt 689 Arg Met Arg Ser Trp Arg Met Asn Val Gly Gly Ser Gly Asp Val Gly ccc aag tat gac agt ggc gag atc ggg ctt acc aag tat gac agt 737 Arg Pro Lys Tyr Asp Ser Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser gag att cct cgg gga tac atc cca tca gtc act aac agc cag atc 785 Gly Glu Ile Pro Arg Gly Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile tca gga gaa atc cct ggt gct tcc cct gac cat cat atg atg tcc cca 833 Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His His Met Met Ser Pro ggg aac att ggc aag cgt gct cca ttt ccc tat gtg aac cat tcg 88ly Asn Ile Gly Lys Arg Ala Pro Phe Pro Tyr Val Asn His Ser aat ccg tca agg gag ttc tct ggt agc att ggg aat gtt gcc tgg 929 Pro Asn Pro Ser Arg Glu Phe Ser Gly Ser Ile Gly Asn Val Ala Trp 2gag agg gtt gat ggc tgg aaa atg aag cag gac aag ggg acg att 977 Lys Glu Arg Val Asp Gly Trp Lys Met Lys Gln Asp Lys Gly Thr Ile 22atg acg aat ggc aca agc att gct ccc tct gag ggt cgg ggt gtt o Met Thr Asn Gly Thr Ser Ile Ala Pro Ser Glu Gly Arg Gly Val 223gt gat att gat gca tca act gat tac aac atg gaa gat gcc tta ttg y Asp Ile Asp Ala Ser Thr Asp Tyr Asn Met Glu Asp Ala Leu Leu 245ac gaa act cga cag cct cta tct agg aaa gtt cca ctt cct tcc n Asp Glu Thr Arg Gln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser 255 26cc agg ata aat cca tac agg atg gtc att gtg ctg cga ttg att gtt r Arg Ile Asn Pro Tyr Arg Met Val Ile Val Leu Arg Leu Ile Val 278gc atc ttc ttg cac tac cgt atc aca aat cct gtg cgc aat gca u Ser Ile Phe Leu His Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala 285 29ac cca tta tgg ctt cta tct gtt ata tgt gag atc tgg ttt gct ctt r Pro Leu Trp Leu Leu Ser Val Ile Cys Glu Ile Trp Phe Ala Leu 33tcg tgg ata ttg gat cag ttc cct aag tgg ttt cca atc aac cgg gag r Trp Ile Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu 323ac ctt gat agg ctg gca tta agg tat gac cgg gaa ggt gag cca r Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro 335 34ct cag ttg gct gct gtt gac att ttc gtc agt aca gtc gac cca atg r Gln Leu Ala Ala Val Asp Ile Phe Val Ser Thr Val Asp Pro Met 356ag cct cct ctt gtc act gcc aat acc gtg cta tcc att ctt gct s Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ala 365 37tg gat tac cct gtg gat aag gtc tct tgc tat gta tct gat gat gga l Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly 389ct gcg atg ctg aca ttt gat gca cta gct gag act tca gag ttt gct a Ala Met Leu Thr Phe Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala 44aaa tgg gta cca ttt gtt aag aag tac aac att gaa cct aga gct g Lys Trp Val Pro Phe Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala 4425 cct gaa tgg tac ttc tcc cag aaa att gat tac ttg aag gac aaa gtg o Glu Trp Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val 434ct tca ttt gtt aaa gac cgc cgg gcc atg aag aga gaa tat gaa s Pro Ser Phe Val Lys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu 445 45aa ttc aaa gtt agg gta aat ggc ctt gtt gct aag gca cag aaa gtt u Phe Lys Val Arg Val Asn Gly Leu Val Ala Lys Ala Gln Lys Val 467ct gag gaa gga tgg atc atg caa gat ggc aca cca tgg cca gga aac o Glu Glu Gly Trp Ile Met Gln Asp Gly Thr Pro Trp Pro Gly Asn 489cc agg gac cat cct gga atg att cag gtt ttc ctt ggt cac agt n Thr Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly His Ser 495 5ggt ggc ctt gat act gag ggc aat gag cta ccc cgt ttg gtc tat gtt y Gly Leu Asp Thr Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val 552gt gaa aag cgt cct gga ttc cag cat cac aag aaa gct ggt gcc r Arg Glu Lys Arg Pro Gly Phe Gln His His Lys Lys Ala Gly Ala 525 53tg aat gct ctt gtt cgt gtc tca gct gtg ctt acc aat gga caa tac t Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr 545tg ttg aat ctt gat tgt gat cac tac att aac aac agt aag gct ctc 2 Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Leu 567aa gct atg tgc ttc ctt atg gac cct aac cta gga agg agt gtc 2 Glu Ala Met Cys Phe Leu Met Asp Pro Asn Leu Gly Arg Ser Val 575 58gc tac gtc cag ttt ccc cag aga ttc gat ggc att gac agg aat gat 2 Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg Asn Asp 59tat gcc aac agg aac acc gtg ttt ttc gat att aac ttg aga ggt 2 Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly 66gat ggc atc caa gga cca gtt tat gtc gga act ggc tgt gtt ttc 2225 Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Val Phe 623ac cga aca gct cta tat ggt tat gag ccc cca att aag cag aag aag 2273 Asn Arg Thr Ala Leu Tyr Gly Tyr Glu Pro Pro Ile Lys Gln Lys Lys 645gt ttc ttg tca tca cta tgt ggc ggt agg aag aag gca agc aaa 232ly Phe Leu Ser Ser Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys 655 66ca aag aag ggc tcg gac aag aag aag tcg cag aag cat gtg gac agt 2369 Ser Lys Lys Gly Ser Asp Lys Lys Lys Ser Gln Lys His Val Asp Ser 678tg cca gta ttc aac ctt gaa gat ata gag gag gga gtt gaa ggc 24Val Pro Val Phe Asn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly 685 69ct gga ttt gac gac gag aaa tca ctt ctt atg tct caa atg agc ctg 2465 Ala Gly Phe Asp Asp Glu Lys Ser Leu Leu Met Ser Gln Met Ser Leu 77gag aag aga ttt ggc cag tcc gca gcg ttt gtt gcc tcc act ctg atg 25Lys Arg Phe Gly Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met 723at ggt ggt gtt cct cag tcc gca act ccg gag tct ctt ctg aaa 256yr Gly Gly Val Pro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys 735 74aa gct atc cat gtt ata agc tgt ggc tat gag gac aag act gaa tgg 26Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp 756ct gag atc ggg tgg atc tac ggt tct gtg aca gaa gac att ctc 2657 Gly Thr Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu 765 77cc gga ttc aag atg cac gcg cga ggc tgg cgg tcg atc tac tgc atg
27Gly Phe Lys Met His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met 789cc aag cgg cca gct ttc aag ggg tct gcc ccc atc aat ctt tcg gac 2753 Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp 88ctg aac cag gtg ctc cgg tgg gct ctt ggg tcc gtg gag atc ctc 28Leu Asn Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu 8825 ttc agc cgg cac tgc ccc ctg tgg tac ggc tac gga ggg cgg ctc aag 2849 Phe Ser Arg His Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys 834tg gag aga ttc gcg tac atc aac acc acc atc tac ccg ctc acg 2897 Phe Leu Glu Arg Phe Ala Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr 845 85cc atc ccg ctt ctc atc tac tgc atc ctg ccc gcc atc tgt ctg ctc 2945 Ser Ile Pro Leu Leu Ile Tyr Cys Ile Leu Pro Ala Ile Cys Leu Leu 867cc gga aag ttc atc att cca gag atc agc aac ttc gcc agc atc tgg 2993 Thr Gly Lys Phe Ile Ile Pro Glu Ile Ser Asn Phe Ala Ser Ile Trp 889tc tcc ctc ttc atc tcg atc ttc gcc acg ggc atc ctg gag atg 3 Ile Ser Leu Phe Ile Ser Ile Phe Ala Thr Gly Ile Leu Glu Met 895 9agg tgg agc ggg gtg ggc atc gac gag tgg tgg agg aac gag cag ttc 3 Trp Ser Gly Val Gly Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe 992tg atc ggg ggc atc tcc gcg cac ctc ttc gcc gtg ttc cag ggc 3 Val Ile Gly Gly Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly 925 93tg ctc aag gtg ctg gcc ggc atc gac acc aac ttc acc gtc acc tcc 3 Leu Lys Val Leu Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser 945ag gcc tcg gac gag gac ggc gac ttc gcg gag ctg tac atg ttc aag 3233 Lys Ala Ser Asp Glu Asp Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys 967cg acg ctc ctg atc ccg ccc acc acc atc ctg atc atc aac ctg 328hr Thr Leu Leu Ile Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu 975 98tc ggc gtc gtc gcc ggc atc tcc tac gcc atc aac agc gga tac cag 3329 Val Gly Val Val Ala Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln 99 tgg ggc ccg ctc ttc ggc aag ctc ttc ttc gcc ttc tgg gtc atc 3377 Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile gtc cac ctg tac ccg ttc ctc aag ggc ctc atg ggc agg cag aac cgc 3425 Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg 25 35 acc ccg acc atc gtc gtc gtc tgg gccatcctgc tggcgtccat cttctccttg 3479 Thr Pro Thr Ile Val Val Val Trp gtgggttc gcatcgaccc cttcaccacc cgcgtcactg gcccggatac ccagacgtgt 3539 ggcatcaact gctagggaag tggaaggttt gtactttgta gaaacggagg aataccacgt 3599 gccatctgtt gtctgttaag ttatatatat ataagcagca agtggcgtta tttacagcta 3659 cgtacagacc agtggatatt gtttaccaca aagttttact tgtgttaata tgcattcttt 37gatata aaaaaaaaaa aaaaaaa 3746 5PRT Zea mays 5lu Gly Asp Ala Asp Gly Val Lys Ser Gly Arg Arg Gly Gly Gly Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly 2 Asp Val Phe Ala Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro 35 4s Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys 5 Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Ala Ile Arg Gly Glu 65 7 Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe Asn Tyr Leu Ala Ser 85 9y Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp Arg Met Arg Ser Trp Met Asn Val Gly Gly Ser Gly Asp Val Gly Arg Pro Lys Tyr Asp Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser Gly Glu Ile Pro Arg Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His His Met Met Ser Pro Thr Gly Asn Ile Gly Arg Ala Pro Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg Phe Ser Gly Ser Ile Gly Asn Val Ala Trp Lys Glu Arg Val Asp 2Trp Lys Met Lys Gln Asp Lys Gly Thr Ile Pro Met Thr Asn Gly 222er Ile Ala Pro Ser Glu Gly Arg Gly Val Gly Asp Ile Asp Ala 225 234hr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg 245 25ln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser Ser Arg Ile Asn Pro 267rg Met Val Ile Val Leu Arg Leu Ile Val Leu Ser Ile Phe Leu 275 28is Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu Trp Leu 29Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp 33Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg 325 33eu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Ala 345sp Ile Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro Leu 355 36al Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val 378ys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr 385 39Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp Val Pro 44Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe 423ln Lys Ile Asp Tyr Leu Lys Asp Lys Val His Pro Ser Phe Val 435 44ys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg 456sn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp 465 478et Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His 485 49ro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr 55Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 5525 Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val 534al Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp 545 556sp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys 565 57he Leu Met Asp Pro Asn Leu Gly Arg Ser Val Cys Tyr Val Gln Phe 589ln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg 595 6Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln 662ro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu 625 634ly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe Leu Ser 645 65er Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys Ser Lys Lys Gly Ser 667ys Lys Lys Ser Gln Lys His Val Asp Ser Ser Val Pro Val Phe 675 68sn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe Asp Asp 69Lys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg Phe Gly 77Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val 725 73ro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val 745er Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly 755 76rp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 778la Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala 785 79Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val 88Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Phe Ser Arg His Cys 823eu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe 835 84la Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu 856yr Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile 865 878ro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser Leu Phe 885 89le Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val 99Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly 9925 Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu 934ly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu 945 956ly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu 965 97le Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala 989le Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu 995 Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val 3l Val Trp 5A Zea mays 5gggcg acgcggacgg cgtga 25 52 25 DNA Zea mays 52 ctagcagttg atgccacacg tctgg 25 53 3753 DNA Zea mays CDS ((34cagcagcaga agcactgcgc ggcattgcag cgatcgagcg ggaggaattt ggggcatggt 6ccaac gccgctcgga tctagaggcc cgcacgggcc gattggtctc cgcccgcctc ggtgttg gtgtcgttgg cgtgtggagc cgtctcggtg ggagcagcgg ggagggagcg atg gcg gcc aac aag ggg atg gtg gcg ggc tcg cac aac cgc aac 228 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn ttc gtc atg atc cgc cac gac ggc gat gtg ccg ggc tcg gct aag 276 Glu Phe Val Met Ile Arg His Asp Gly Asp Val Pro Gly Ser Ala Lys 2 ccc aca aag agt gcg aat gga cag gtc tgc cag att tgc ggt gac tct 324 Pro Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Ser 35 4g ggt gtt tca gcc act ggt gat gtc ttt gtt gcc tgc aat gag tgt 372 Val Gly Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys 5 gcc ttc cct gtc tgc cgc cca tgc tat gag tat gag cgc aag gag ggg 42he Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly 65 7c caa tgc tgc ccc cag tgc aag act aga tac aag aga cag aaa ggt 468 Asn Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly 8 95 agc cct cga gtt cat ggt gat gag gat gag gaa gat gtt gat gac cta 5Pro Arg Val His Gly Asp Glu Asp Glu Glu Asp Val Asp Asp Leu aat gaa ttc aac tac aag caa ggc agt ggg aaa ggc cca gag tgg 564 Asp Asn Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys Gly Pro Glu Trp ctg caa gga gat gat gct gat ctg tct tca tct gct cgc cat gag 6Leu Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Glu cat cat cgg att cca cgc ctg aca agc ggt caa cag ata tct gga 66is His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly att cct gat gct tcc cct gac cgt cat tct atc cgc agt cca aca 7Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr tcg agc tat gtt gat cca agc gtc cca gtt cct gtg agg att gtg gac 756 Ser Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp tcg aag gac ttg aat tcc tat ggg ctt aat agt gtt gac tgg aag 8Ser Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys 2aga gtt gag agc tgg agg gtt aaa cag gac aaa aat atg atg caa 852 Glu Arg Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Met Gln 222ct aat aaa tat cca gag gct aga gga gga gac atg gag ggg act 9Thr Asn Lys Tyr Pro Glu Ala Arg Gly Gly Asp Met Glu Gly Thr 225 23gc tca aat gga gaa gat atg caa atg gtt gat gat gca cgg cta cct 948 Gly Ser Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro 245tg agc cgt atc gtg cca att tcc tca aac cag ctc aac ctt tac cgg 996 Leu Ser Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg 267tg atc att ctc cgt ctt atc atc ctg tgc ttc ttc ttc cag tat l Val Ile Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr 275 28gt gtc agt cat cca gtg cgt gat gct tat gga tta tgg cta gta tct g Val Ser His Pro Val Arg Asp Ala Tyr Gly Leu Trp Leu Val Ser 29atc tgc gag gtc tgg ttt gcc ttg tct tgg ctt cta gat cag ttc l Ile Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe 33aaa tgg tat cca atc aac cgt gag aca tat ctt gac agg ctt gca o Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala 323tg agg tat gat aga gag gga gag cca tca cag ctg gct ccc att gat u Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp 345tc gtc agt aca gtg gat cca ttg aag gaa cct cca ctg atc aca l Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr 355 36cc aac act gtt ttg tcc att ctt tct gtg gat tac cct gtt gac aaa a Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp Lys 378ca tgc tat gtt tct gat gat ggt tca gct atg ctg act ttt gag l Ser Cys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu 385 39ct ctc tca gaa acc gca gaa ttt gct aga aag tgg gtt ccc ttt tgt r Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys 44aag aag cac aat att gaa cca aga gct cca gaa ttt tac ttt gct caa s Lys His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln 423ta gat tac ctg aag gac aaa att caa cct tca ttt gtt aag gaa s Ile Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu 435 44ga cgc gca atg aag agg gag tat gaa gaa ttc aaa gta aga atc aat g Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn 456tt gtt gcc aaa gca cag aaa gtg cct gaa gag ggg tgg acc atg a Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met 465 47ct gat gga act gca tgg cct ggg aat aat cct agg gac cat cct ggc a Asp Gly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly 489tg att cag gtt ttc ttg ggg cac agt ggt ggg ctc gac act gat gga t Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly 55gag tta cca cgt ctt gtc tat gtc tct cgt gaa aag aga cca ggc n Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly 5525 ttt cag cat cac aag aag gct ggt gca atg aat gcg ctg att cgt gta e Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile
Arg Val 534ct gtg ctg aca aat ggt gcc tat ctt ctc aat gtg gat tgc gac r Ala Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp 545 55at tac ttc aat agc agc aaa gct ctt aga gaa gca atg tgc ttc atg s Tyr Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met 567tg gat ccg gct cta gga agg aaa act tgt tat gta caa ttt cca cag t Asp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln 589tt gat ggc att gac ttg cac gat cga tat gct aat cgg aac ata 2 Phe Asp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile 595 6gtt ttc ttt gat atc aac atg aaa ggt ctg gat ggc att cag ggt cca 2 Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro 662ac gtg gga aca gga tgc tgt ttc aat aga cag gct ttg tat gga 2 Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly 625 63ac gat cct gtt ttg act gaa gct gat ctg gag cca aac att gtt att 2 Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Ile 645ag agc tgc tgt ggt aga agg aag aaa aag aac aag agt tat atg gat 2 Ser Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys Ser Tyr Met Asp 667aa agc cgt att atg aag aga aca gaa tct tca gct ccc atc ttc 2244 Ser Gln Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe 675 68at atg gaa gac atc gaa gag ggt att gaa ggt tac gag gat gaa agg 2292 Asn Met Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg 69gtg ctt atg tcc cag agg aaa ttg gag aaa cgc ttt ggt cag tct 234al Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser 77att ttc att gca tcc acc ttt atg aca caa ggt ggc ata cca cct 2388 Pro Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro 723ca aca aac cca gct tct cta cta aag gaa gct atc cat gtc atc agt 2436 Ser Thr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser 745ga tat gag gac aaa act gaa tgg gga aaa gag att ggc tgg atc 2484 Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile 755 76at ggt tca gta acg gag gat att ctg act ggg ttt aaa atg cat gca 2532 Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala 778gc tgg caa tca atc tac tgc atg cca cca cga cct tgt ttc aag 258ly Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys 785 79gt tct gca cca atc aat ctt tcc gat cgt ctt aat cag gtg ctc cgt 2628 Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg 88tgg gct ctt ggg tca gtg gaa att ctg ctt agt aga cat tgt cct atc 2676 Trp Ala Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile 823at ggt tac aat gga cga ttg aag ctt ttg gag agg ctg gct tac 2724 Trp Tyr Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr 835 84tc aac act att gta tat cca atc aca tcc att ccg ctt att gcc tat 2772 Ile Asn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Ile Ala Tyr 856tg ctt ccc gct atc tgc ctc ctt acc aat aaa ttt atc att cct 282al Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro 865 87ag att agc aat tat gct ggg atg ttc ttc att ctt ctt ttc gcc tcc 2868 Glu Ile Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser 889tt ttt gcc act ggt ata ttg gag ctt aga tgg agt ggt gtt ggc att 29Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile 99gat tgg tgg aga aat gag cag ttt tgg gtt att ggt ggc acc tct 2964 Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser 9925 gcc cat ctc ttc gca gtg ttc cag ggt ctg ctg aaa gtg ttg gct ggg 3 His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly 934at acc aac ttc aca gtt acc tca aag gca tct gat gag gat ggc 3 Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly 945 95ac ttt gct gag cta tat gtg ttc aag tgg acc agt ttg ctc att cct 3 Phe Ala Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro 967cg acc act gtt ctt gtc att aac ctg gtc gga atg gtg gca gga att 3 Thr Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile 989at gcc att aac agt ggc tac caa tcc tgg ggt ccg ctc ttt gga 32Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly 995 ctg ttc ttc tcg atc tgg gtg atc ctc cat ctc tac ccc ttc ctc 3252 Lys Leu Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu aag ggt ctc atg gga agg cag aac cgc aca cca aca atc gtc att gtc 33Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val 3tgg tcc atc ctt ctt gca tct atc ttc tcc ttg ctg tgg gtg aag atc 3348 Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile 45 55 gat cct ttc atc tcc ccg aca cag aaa gct gct gcc ttg ggg caa tgt 3396 Asp Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys 65 c gtc aac tgc tgatcgagac agtgactctt atttgaagag gctcaatcaa 3448 Gly Val Asn Cys tctgcccc ctcgtgtaaa tacctgagga ggctagatgg gaattccttt tgttgtaggt 35atggat ttgcatctaa gttatgcctc tgttcattag cttcttccgt gccggtgctg 3568 ctgcggacta agaatcacgg agcctttcta ccttccatgt agcgccagcc agcagcgtaa 3628 gatgtgaatt ttgaagtttt gttatgcgtg cagtttattg ttttagagta aattatcatt 3688 tgtttgtggg aactgttcac acgagcttat aatggcaatg ctgttattta aaaaaaaaaa 3748 aaaaa 3753 54 T Zea mays 54 Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu Val Met Ile Arg His Asp Gly Asp Val Pro Gly Ser Ala Lys Pro 2 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Ser Val 35 4y Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 5 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn 65 7 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 9o Arg Val His Gly Asp Glu Asp Glu Glu Asp Val Asp Asp Leu Asp Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys Gly Pro Glu Trp Gln Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Glu Pro His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 2Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Met Gln Val 222sn Lys Tyr Pro Glu Ala Arg Gly Gly Asp Met Glu Gly Thr Gly 225 234sn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu 245 25er Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Val 267le Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg 275 28al Ser His Pro Val Arg Asp Ala Tyr Gly Leu Trp Leu Val Ser Val 29Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro 33Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu 325 33rg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val 345al Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala 355 36sn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp Lys Val 378ys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser 385 39Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys 44His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys 423sp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg 435 44rg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala 456al Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala 465 478ly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met 485 49le Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn 55Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe 5525 Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser 534al Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His 545 556he Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met 565 57sp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg 589sp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val 595 6Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val 662al Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr 625 634ro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Ile Lys 645 65er Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys Ser Tyr Met Asp Ser 667er Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn 675 68et Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser 69Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro 77Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser 725 73hr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys 745yr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr 755 76ly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg 778rp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly 785 79Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp 88Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp 823ly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile 835 84sn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Ile Ala Tyr Cys 856eu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu 865 878er Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile 885 89he Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu 99Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala 9925 His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile 934hr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp 945 956la Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro 965 97hr Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser 989la Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys 995 Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp 3r Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp 5Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly 65 l Asn Cys 25 DNA Zea mays 55 atggcggcca acaaggggat ggtgg 25 56 25 DNA Zea mays 56 tcagcagttg acgccacatt gcccc 25 57 37Zea mays CDS (272)...(3499) 57 gtcgacccac gcttccggtc ggttccgcgt cccttttccc ctcccccctc cgtcgccgcc 6cgagc tccaccactt gctcctgcgc gaggtgaaca ctgggttagg gccactgcca ctgggct gcctctgctt ctgcctctcc cgccagcgcg cgagcccggg ggcgattcgg cggcacg cgggagggga agccgaggaa tgcggtgagt cggcgggggt ccggcgtttg 24tcgtg gagggctcgg attggtgcgc c atg gac ggc ggc gac gcc acg 292 Met Asp Gly Gly Asp Ala Thr tcg ggg aag cat gtg gcc ggg cag gtg tgc cag atc tgc ggc gac 34er Gly Lys His Val Ala Gly Gln Val Cys Gln Ile Cys Gly Asp tg ggc acc gcg gcg gac ggc gac ctc ttc acc gcc tgc gac gtc 388 Gly Val Gly Thr Ala Ala Asp Gly Asp Leu Phe Thr Ala Cys Asp Val 25 3c ggc ttc ccc gtg tgc cgc cca tgc tac gag tac gag cgc aag gac 436 Cys Gly Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Asp 4 55 ggc acc cag gcg tgc ccg cag tgc aag act aag tac aag cgc cac aaa 484 Gly Thr Gln Ala Cys Pro Gln Cys Lys Thr Lys Tyr Lys Arg His Lys 6 ggg agc cca cca gta cac ggt gag gaa aat gag gat gtg gat gct gac 532 Gly Ser Pro Pro Val His Gly Glu Glu Asn Glu Asp Val Asp Ala Asp 75 8t gtg agt gac tac aac tac caa gca tct ggc aac cag gat cag aag 58al Ser Asp Tyr Asn Tyr Gln Ala Ser Gly Asn Gln Asp Gln Lys 9ag att gct gag aga atg ctc act tgg cgg aca aac tca cgt ggc 628 Gln Lys Ile Ala Glu Arg Met Leu Thr Trp Arg Thr Asn Ser Arg Gly gat att ggc ctg gct aag tat gac agc ggt gaa att ggg cat ggg 676 Ser Asp Ile Gly Leu Ala Lys Tyr Asp Ser Gly Glu Ile Gly His Gly aag tat gac agt ggt gag atc cct cgt gga tat atc ccg tca cta act 724 Lys Tyr Asp Ser Gly Glu Ile Pro Arg Gly Tyr Ile Pro Ser Leu Thr agc cag atc tca gga gag att cct gga gct tcc cct gat cat atg 772 His Ser Gln Ile Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His Met tct cct gtt ggg aac att ggc agg cgt gga cat caa ttt cct tat 82er Pro Val Gly Asn Ile Gly Arg Arg Gly His Gln Phe Pro Tyr aat cat tct cca aac cca tcg agg gag ttc tcc ggt agc ctt ggc 868 Val Asn His Ser Pro Asn Pro Ser Arg Glu Phe Ser Gly Ser Leu Gly gtt gca tgg aaa gag agg gtg gat gga tgg aaa atg aag gat aaa 9Val Ala Trp Lys Glu Arg Val Asp Gly Trp Lys Met Lys Asp Lys 22ggt gca att cct atg acc aat gga aca agc att gct cca tca
gaa ggg 964 Gly Ala Ile Pro Met Thr Asn Gly Thr Ser Ile Ala Pro Ser Glu Gly 223ga gtt gct gat att gat gct tct act gat tat aac atg gaa gat g Gly Val Ala Asp Ile Asp Ala Ser Thr Asp Tyr Asn Met Glu Asp 235 24cc tta ctg aat gat gaa act cgg caa cct cta tct aga aaa gtg cca a Leu Leu Asn Asp Glu Thr Arg Gln Pro Leu Ser Arg Lys Val Pro 256ct tca tcc aga ata aat ccg tac aga atg gtc att gtg cta cgt e Pro Ser Ser Arg Ile Asn Pro Tyr Arg Met Val Ile Val Leu Arg 265 27tg gct gtt cta tgc ata ttc ttg cgc tac cgt atc aca cat cct gtg u Ala Val Leu Cys Ile Phe Leu Arg Tyr Arg Ile Thr His Pro Val 289ac aat gca tat cca ctg tgg ctt tta tcc gtc ata tgt gag atc tgg n Asn Ala Tyr Pro Leu Trp Leu Leu Ser Val Ile Cys Glu Ile Trp 33gct ttg tcc tgg att ttg gat cag ttc cca aag tgg tcc cca atc e Ala Leu Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Ser Pro Ile 3325 aac cgt gaa aca tac ctt gat aga ctg gct tta agg tat gac cga gaa n Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu 334aa cca tct caa tta gct cct gtt gat att ttt gtc agt act gtg y Glu Pro Ser Gln Leu Ala Pro Val Asp Ile Phe Val Ser Thr Val 345 35at cca atg aag gag cct cct ctt gtc act gca aat act gtg ctt tcc p Pro Met Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu Ser 367tc ctt gct gtc gat tat ccg gtt gac aag gta tct tgc tat gtt tcg e Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val Ser 389at gga gct gct atg ctg act ttt gat gct ctc tct gaa act tca p Asp Gly Ala Ala Met Leu Thr Phe Asp Ala Leu Ser Glu Thr Ser 395 4gag ttt gct aga aaa tgg gtt ccg ttc tgt aag aag tac aac ata gag u Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Tyr Asn Ile Glu 442gg gcc ccg gaa tgg tac ttt gct cag aaa att gat tac ttg aaa o Arg Ala Pro Glu Trp Tyr Phe Ala Gln Lys Ile Asp Tyr Leu Lys 425 43ac aaa gtt caa acc tca ttt gtg aaa gaa cgc cgg gcc atg aag aga p Lys Val Gln Thr Ser Phe Val Lys Glu Arg Arg Ala Met Lys Arg 445aa tat gaa gaa ttc aaa gtt cgt atc aat ggt ctt gta gcc aag gca u Tyr Glu Glu Phe Lys Val Arg Ile Asn Gly Leu Val Ala Lys Ala 467aa gtt ccc gag gag gga tgg atc atg caa gat ggt aca cct tgg n Lys Val Pro Glu Glu Gly Trp Ile Met Gln Asp Gly Thr Pro Trp 475 48ct ggg aac aat act agg gac cat cct gga atg att cag gtt ttc ctg o Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val Phe Leu 49cac agt gga ggg ctt gac gtt gaa ggc aat gaa ctt cct cgt ttg y His Ser Gly Gly Leu Asp Val Glu Gly Asn Glu Leu Pro Arg Leu 55tat gtg tct cgt gaa aaa cgt cct gga ttc caa cat cac aag aag l Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His Lys Lys 523ct ggt gcc atg aat gca ctt gtt cgt gta tca gct gtc ctt act aat a Gly Ala Met Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn 545aa tac atg ttg aat ctt gat tgt gac cac tac atc aat aat agc y Gln Tyr Met Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser 555 56ag gct ctt cga gaa gct atg tgc ttc ctt atg gac cca aac cta gga 2 Ala Leu Arg Glu Ala Met Cys Phe Leu Met Asp Pro Asn Leu Gly 578at gtc tgt tat gtc caa ttt cct cag agg ttt gat ggt att gat 2 Asn Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp 585 59gg aat gac cga tat gca aac agg aac act gtg ttt ttc gat att aac 2 Asn Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp Ile Asn 66ttg aga ggt ctt gac ggc att caa ggg cca gtt tat gtg gga act ggt 2 Arg Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr Gly 623tg ttt aac aga acg gcc tta tat ggt tat gag cct cca gtc aag 22Val Phe Asn Arg Thr Ala Leu Tyr Gly Tyr Glu Pro Pro Val Lys 635 64aa aaa aag cca ggc ttc ttc tct tcg ctt tgt ggg gga agg aaa aag 226ys Lys Pro Gly Phe Phe Ser Ser Leu Cys Gly Gly Arg Lys Lys 656ca aaa tct aag aag agc tcg gaa aag aag aag tca cat aga cac 23Ser Lys Ser Lys Lys Ser Ser Glu Lys Lys Lys Ser His Arg His 665 67ca gac agt tct gta cca gta ttt aat ctc gaa gat ata gag gaa ggg 2356 Ala Asp Ser Ser Val Pro Val Phe Asn Leu Glu Asp Ile Glu Glu Gly 689tt gaa ggt tct cag ttt gat gat gag aaa tcg ctg att atg tct caa 24Glu Gly Ser Gln Phe Asp Asp Glu Lys Ser Leu Ile Met Ser Gln 77agc ttg gag aag aga ttt ggc cag tcc agt gtt ttt gta gcc tct 2452 Met Ser Leu Glu Lys Arg Phe Gly Gln Ser Ser Val Phe Val Ala Ser 7725 act ctg atg gaa tat ggt ggt gtt cca caa tct gca act cca gag tct 25Leu Met Glu Tyr Gly Gly Val Pro Gln Ser Ala Thr Pro Glu Ser 734tg aaa gaa gct att cat gtc atc agc tgt ggc tat gag gac aaa 2548 Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp Lys 745 75ct gac tgg gga act gag att ggg tgg atc tat ggt tct gtt aca gaa 2596 Thr Asp Trp Gly Thr Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu 767ac att ctc acc gga ttc aag atg cat gct cga ggc tgg cga tca atc 2644 Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly Trp Arg Ser Ile 789gc atg cct aag cga cca gct ttc aag gga tct gct cct atc aac 2692 Tyr Cys Met Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn 795 8ctt tcg gat cgt ttg aat caa gtg ctt cgg tgg gct ctt ggt tcc att 274er Asp Arg Leu Asn Gln Val Leu Arg Trp Ala Leu Gly Ser Ile 882tt ctt ttc agc agg cat tgt ccc ata tgg tat ggc tat gga ggc 2788 Glu Ile Leu Phe Ser Arg His Cys Pro Ile Trp Tyr Gly Tyr Gly Gly 825 83gg ctt aaa ttc ctg gag aga ttt gct tat atc aac aca aca att tat 2836 Arg Leu Lys Phe Leu Glu Arg Phe Ala Tyr Ile Asn Thr Thr Ile Tyr 845ca ctc aca tca atc ccg ctc ctc ctg tac tgc ata ttg cca gca gtt 2884 Pro Leu Thr Ser Ile Pro Leu Leu Leu Tyr Cys Ile Leu Pro Ala Val 867tt ctc act ggg aag ttc atc atc cca aag att agt aac cta gag 2932 Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Lys Ile Ser Asn Leu Glu 875 88gt gtt tgg ttt ata tcg ctc ttt atc tca atc ttt gcc act ggt atc 298al Trp Phe Ile Ser Leu Phe Ile Ser Ile Phe Ala Thr Gly Ile 89gag atg agg tgg agt ggt gtt ggc att gat gaa tgg tgg agg aac 3 Glu Met Arg Trp Ser Gly Val Gly Ile Asp Glu Trp Trp Arg Asn 99cag ttc tgg gtc att ggt ggt att tct gcg cat tta ttt gcc gtc 3 Gln Phe Trp Val Ile Gly Gly Ile Ser Ala His Leu Phe Ala Val 923tc cag ggt ctc ctg aag gtg ctt gct ggt atc gac acg agc ttc act 3 Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp Thr Ser Phe Thr 945cc tct aag gcc act gac gaa gaa ggt gat ttt gcc gag ctc tac 3 Thr Ser Lys Ala Thr Asp Glu Glu Gly Asp Phe Ala Glu Leu Tyr 955 96tg ttc aag tgg aca acg ctt ctg atc cca cca acc act att ttg atc 322he Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Ile Leu Ile 978ac ctg gtc ggc gtg gtc gct ggc att tcc tac gca atc aat agc 3268 Ile Asn Leu Val Gly Val Val Ala Gly Ile Ser Tyr Ala Ile Asn Ser 985 99gt tac cag tca tgg gga cct ctt ttc ggg aag ctc ttc ttt gcg ttc 33Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe g gtg att gtc cac ctg tac ccc ttc ctc aag ggc ctc atg ggg aag 3364 Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Lys 25 g aac cgc acg ccg acc att gtc gtt gtc tgg gct atc ctc ctt gcg 34Asn Arg Thr Pro Thr Ile Val Val Val Trp Ala Ile Leu Leu Ala 4tcg atc ttt tcc ctg atg tgg gtt cgt atc gat cca ttc acc acc cgg 346le Phe Ser Leu Met Trp Val Arg Ile Asp Pro Phe Thr Thr Arg 55 c act ggc cct gat atc gcg aaa tgt ggc atc aac tgc taggatgagc 35Thr Gly Pro Asp Ile Ala Lys Cys Gly Ile Asn Cys 7tgaagatagt taaagagtgg aactagacgc attgtgcatc gtaagttatc agtgggtggc 3569 tctttttata gtatggtagg aacttggtcg ggagacgtta attacatatg ctatatgtac 3629 ctccgctggt ctttatccgt aagttaatat atatactgct ttgagaatta aaaaaaaaaa 3689 aaaaagggcg gccgc 37 Zea mays 58 Met Asp Gly Gly Asp Ala Thr Asn Ser Gly Lys His Val Ala Gly Gln Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Ala Ala Asp Gly Asp 2 Leu Phe Thr Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro Cys 35 4r Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys Lys 5 Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Pro Val His Gly Glu Glu 65 7 Asn Glu Asp Val Asp Ala Asp Asp Val Ser Asp Tyr Asn Tyr Gln Ala 85 9r Gly Asn Gln Asp Gln Lys Gln Lys Ile Ala Glu Arg Met Leu Thr Arg Thr Asn Ser Arg Gly Ser Asp Ile Gly Leu Ala Lys Tyr Asp Gly Glu Ile Gly His Gly Lys Tyr Asp Ser Gly Glu Ile Pro Arg Tyr Ile Pro Ser Leu Thr His Ser Gln Ile Ser Gly Glu Ile Pro Gly Ala Ser Pro Asp His Met Met Ser Pro Val Gly Asn Ile Gly Arg Gly His Gln Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg Phe Ser Gly Ser Leu Gly Asn Val Ala Trp Lys Glu Arg Val Asp 2Trp Lys Met Lys Asp Lys Gly Ala Ile Pro Met Thr Asn Gly Thr 222le Ala Pro Ser Glu Gly Arg Gly Val Ala Asp Ile Asp Ala Ser 225 234sp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg Gln 245 25ro Leu Ser Arg Lys Val Pro Ile Pro Ser Ser Arg Ile Asn Pro Tyr 267et Val Ile Val Leu Arg Leu Ala Val Leu Cys Ile Phe Leu Arg 275 28yr Arg Ile Thr His Pro Val Asn Asn Ala Tyr Pro Leu Trp Leu Leu 29Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp Gln 33Phe Pro Lys Trp Ser Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu 325 33la Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Val 345le Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro Leu Val 355 36hr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp 378al Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe 385 39Ala Leu Ser Glu Thr Ser Glu Phe Ala Arg Lys Trp Val Pro Phe 44Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Ala 423ys Ile Asp Tyr Leu Lys Asp Lys Val Gln Thr Ser Phe Val Lys 435 44lu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile 456ly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Ile 465 478ln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro 485 49ly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Val Glu 55Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro 5525 Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg 534er Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp Cys 545 556is Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys Phe 565 57eu Met Asp Pro Asn Leu Gly Arg Asn Val Cys Tyr Val Gln Phe Pro 589rg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg Asn 595 6Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln Gly 662al Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu Tyr 625 634yr Glu Pro Pro Val Lys Lys Lys Lys Pro Gly Phe Phe Ser Ser 645 65eu Cys Gly Gly Arg Lys Lys Thr Ser Lys Ser Lys Lys Ser Ser Glu 667ys Lys Ser His Arg His Ala Asp Ser Ser Val Pro Val Phe Asn 675 68eu Glu Asp Ile Glu Glu Gly Ile Glu Gly Ser Gln Phe Asp Asp Glu 69Ser Leu Ile Met Ser Gln Met Ser Leu Glu Lys Arg Phe Gly Gln 77Ser Ser Val Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val Pro 725 73ln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val Ile 745ys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Thr Glu Ile Gly Trp 755 76le Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His 778rg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala Phe 785 79Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu 88Trp Ala Leu Gly Ser Ile Glu Ile Leu Phe Ser Arg His Cys Pro 823rp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe Ala 835 84yr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu Leu 856ys Ile Leu Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile 865 878ys Ile Ser Asn Leu Glu Ser Val Trp Phe Ile Ser Leu Phe Ile 885 89er Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val Gly 99Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Ile 9925 Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala 934le Asp Thr Ser Phe Thr Val Thr Ser Lys Ala Thr Asp Glu Glu 945 956sp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu Ile 965 97ro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala Gly 989er Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe 995 Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu
Lys Gly Leu Met Gly Lys Gln Asn Arg Thr Pro Thr Ile Val Val 3l Trp Ala Ile Leu Leu Ala Ser Ile Phe Ser Leu Met Trp Val Arg 5Ile Asp Pro Phe Thr Thr Arg Val Thr Gly Pro Asp Ile Ala Lys Cys 65 y Ile Asn Cys 25 DNA Zea mays 59 atggacggcg gcgacgccac gaatt 25 6A Zea mays 6agttg atgccacatt tcgcg 25 6A Artificial Sequence oligonucleotide probe 6ccacg cgtccgaaaa aaaaaaaaaa aaaaaa 36
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